_:b444951987 . _:b444952158 "2"^^ . _:b444952153 "2"^^ . _:b444952152 "2"^^ . . . _:b444952155 "2"^^ . _:b444952154 "2"^^ . _:b444952165 "2"^^ . _:b444952065 . _:b8944684 . _:b444952164 "2"^^ . _:b444952167 "2"^^ . _:b444952166 "2"^^ . _:b444952161 "2"^^ . . _:b444952160 "2"^^ . _:b444951995 . _:b444952163 "2"^^ . _:b444952001 . . _:b444952162 "2"^^ . _:b444951933 . _:b444952173 "2"^^ . . _:b444952161 . _:b444952172 "2"^^ . _:b8944685 "GST pull-down experiments were performed as described previously (Bienko et al., >>2005<<) using proteins obtained as follows: GST, GST-PCNA, His6-PCNA, and GST-FANCL (amino acids 1\u2013306) were purified from E. coli using glutathione agarose (Thermo Fisher Scientific) or TALON Superflow metal affinity resin (Takara Bio Inc." . _:b444952175 "2"^^ . _:b444952174 "2"^^ . _:b444952224 . _:b444952169 "2"^^ . _:b8944657 "of the ID complex targets it to the stalled replication fork or site of damage, where it promotes processing and repair of the ICL lesion (R\u00E4schle et al., 2008; Knipscheer et al., 2009; Kratz et al., 2010; Smogorzewska et al., >>2010<<)." . _:b444952168 "2"^^ . _:b444952171 "2"^^ . _:b444951969 . _:b444952158 . _:b444952170 "2"^^ . _:b444952181 "2"^^ . _:b444951917 . _:b444952180 "2"^^ . . _:b444952183 "2"^^ . _:b8944658 . _:b444952100 . _:b444951919 . _:b444952182 "2"^^ . . _:b444951918 . _:b444952177 "2"^^ . _:b444952176 "2"^^ . _:b444952179 "2"^^ . . . _:b444952131 . _:b444952178 "2"^^ . _:b444952018 . . _:b444952189 "2"^^ . _:b444951925 . _:b444952188 "2"^^ . _:b444951924 . _:b444952191 "2"^^ . _:b8944698 . . _:b444952190 "2"^^ . _:b444951927 . _:b444951926 . _:b444952185 "2"^^ . _:b444951976 . . _:b444951921 . _:b444952184 "2"^^ . _:b444952034 . _:b444951920 . _:b444952187 "2"^^ . _:b8944674 "Wang, National Institutes of Health, Bethesda, MD), ORC2 (BD), \u03B2-actin (Sigma-Aldrich), HA tag (Covance), S tag (Hackbarth et al., >>2004<<), and Flag tag (Flag M1 mAb; Sigma-Aldrich)." . _:b444951923 . _:b444952186 "2"^^ . _:b444951922 . _:b444952197 "2"^^ . . _:b444951933 . _:b444952196 "2"^^ . . _:b444951932 . _:b444952199 "2"^^ . _:b444951935 . _:b444952198 "2"^^ . _:b444952133 . _:b444951934 . _:b444952193 "2"^^ . . _:b444951929 . _:b444952192 "2"^^ . . _:b444951928 . _:b8944673 "Shiomi (National Institute of Radiological Sciences, Chiba, Japan) and grown as described previously (Shiomi et al., >>2007<<)." . _:b444952195 "2"^^ . _:b444952038 . . _:b444951931 . _:b444952194 "2"^^ . . _:b444952108 . . _:b444952037 . _:b444951930 . _:b444952214 . _:b444952205 "2"^^ . _:b444952184 . _:b444951941 . _:b444952204 "2"^^ . . _:b444952159 . _:b8944678 "5\u2032-CUUACGCUGAGUACUUCGA-3\u2032 (Elbashir et al., 2001); FANCD2, 5\u2032-GGUCAGAGCUGUAUUAUUC-3\u2032 (Wagner and Karnitz, 2009); PCNA, 5\u2032-GGAGAAAGUUUCAGACUAU-3\u2032 (Merkerova et al., 2007); and FANCL, 5\u2032-GACAAGAGCUGUAUGCACU-3\u2032 (Meetei et al., >>2003<<)." . _:b444951940 . _:b444952207 "2"^^ . _:b444951943 . _:b444952206 "2"^^ . _:b444952096 . _:b444951942 . _:b444952201 "2"^^ . _:b444951937 . _:b444952200 "2"^^ . . _:b444951949 . _:b444951936 . _:b444952203 "2"^^ . _:b444952228 . _:b444951939 . _:b444952202 "2"^^ . _:b444952224 . _:b444952225 . _:b444952213 "2"^^ . _:b444951938 . _:b444952226 . _:b444952039 . _:b444952227 . _:b444952054 . . _:b444952212 "2"^^ . _:b444951949 . _:b444951948 . _:b444952215 "2"^^ . . _:b444951979 . _:b444952212 . _:b8944692 "that both RAD18 and FANCD2 play important roles in eukaryotic cells exposed to cisplatin and other DNA cross-linking agents (Yamashita et al., 2002; Nojima et al., 2005; Ross et al., 2005; Lee and Myung, 2008; Wagner and Karnitz, >>2009<<; Hicks et al., 2010)." . _:b444952213 . _:b444952214 "2"^^ . _:b444951951 . _:b444952214 . _:b444952215 . _:b444952208 . _:b444952153 . _:b444952209 . _:b444952209 "2"^^ . _:b444951950 . _:b444952210 . _:b444952211 . . _:b444952220 . _:b444952221 . _:b444952208 "2"^^ . _:b444951945 . _:b444952222 . _:b444952223 . . _:b444952216 . _:b444952217 . _:b444952211 "2"^^ . _:b444951944 . _:b444952218 . _:b444952219 . _:b444952196 . _:b8944666 . _:b444952197 . _:b444952210 "2"^^ . _:b444951947 . _:b444952198 . _:b444952113 . _:b444952199 . _:b444952213 . _:b444952062 . _:b444952192 . _:b444952193 . _:b444952221 "2"^^ . _:b444951946 . . _:b444952194 . _:b444952195 . _:b444952204 . _:b444952205 . _:b444952220 "2"^^ . _:b444952156 . _:b444951957 . _:b444952206 . _:b444952207 . _:b444952200 . _:b444952201 . _:b444952223 "2"^^ . _:b444951956 . . _:b444952202 . _:b444952203 . _:b444952180 . _:b444952181 . _:b444952222 "2"^^ . _:b444951959 . _:b444952182 . _:b444952183 . _:b444952176 . _:b444952177 . _:b444952217 "2"^^ . _:b444951958 . _:b444952178 . _:b8944671 "Cell cycle analyses (Arlander et al., >>2003<<) and clonogenic assays (Wagner and Karnitz, 2009) were performed as described previously." . . _:b444952179 . . _:b444952188 . _:b444952189 . _:b444952216 "2"^^ . _:b444951953 . _:b444952190 . _:b444952204 . _:b444952191 . _:b444952184 . _:b444952185 . _:b444952219 "2"^^ . _:b444951952 . _:b444951945 . _:b444952186 . _:b444952187 . _:b444952150 . . _:b444952164 . _:b444952165 . _:b444952218 "2"^^ . _:b444951955 . _:b444952166 . _:b444952167 . _:b444952007 . _:b444952160 . _:b444952161 . _:b8944697 "S1 B), which creates bulky DNA lesions that are bypassed by TLS polymerases after RAD18-mediated PCNA ubiquitylation (Tateishi et al., 2000; Kannouche et al., 2004; Watanabe et al., >>2004<<). Surprisingly, however, codepletion of both proteins did not cause a more profound sensitization to cisplatin. Given that the RAD18 and FANCD2 depletions were both highly effective, this result raised the possibility that these proteins" . _:b444951954 . _:b444952162 . . _:b444952163 . . . _:b444952172 . _:b444952173 . _:b444952228 "2"^^ . _:b444951965 . _:b444952174 . _:b444952175 . _:b444952168 . _:b444952169 . _:b444951964 . _:b444952170 . . _:b444952171 . _:b444952148 . _:b444952149 . _:b444951967 . . _:b444952150 . _:b444952151 . . _:b444952144 . . _:b444952225 "2"^^ . _:b444951966 . _:b444952145 . _:b444952046 . _:b444952146 . _:b444952147 . . _:b444952156 . _:b444952157 . _:b444952224 "2"^^ . _:b444951961 . _:b444952158 . _:b444952159 . _:b444952152 . _:b444952153 . _:b444952227 "2"^^ . _:b444951960 . _:b444952154 . . _:b444952155 . _:b444952132 . _:b444952133 . _:b444952226 "2"^^ . _:b444951963 . _:b444952134 . _:b444952135 . _:b444952128 . _:b444952129 . _:b444951962 . _:b444952130 . . _:b444952131 . _:b444952140 . _:b8944673 . _:b444952004 . _:b444952141 . _:b444951973 . _:b444952142 . . _:b444952143 . _:b444952136 . _:b444952137 . _:b444951972 . _:b444952049 . _:b444952138 . _:b444952139 . _:b444952197 . _:b444952116 . . _:b444952117 . _:b444951975 . . _:b444952118 . _:b444952119 . _:b444952112 . _:b444952113 . _:b444951974 . _:b444952114 . _:b444952115 . _:b444952223 . _:b444952124 . _:b444952125 . _:b444951969 . _:b444952106 . _:b444952126 . . _:b444952127 . . _:b444952120 . _:b444952121 . _:b444952162 . _:b444951968 . _:b444952122 . _:b444952123 . _:b444952040 . _:b444952100 . _:b444952101 . _:b444951971 . _:b444952102 . _:b444952103 . . _:b444952096 . _:b444952097 . _:b444951970 . _:b444952098 . _:b444952099 . _:b444952108 . _:b444952109 . _:b8944655 . _:b444951981 . _:b444952110 . _:b444952111 . _:b8944697 . _:b444952104 . . _:b444951980 . _:b444952105 . _:b444952106 . . _:b444952107 . _:b444952084 . _:b444952031 . _:b444951983 . _:b444952085 . _:b444952086 . . _:b444952087 . . . _:b444952080 . . _:b8944676 "RAD18-4, 5\u2032-AUAACCGCAUAUUAGAUGA-3\u2032; RAD18-5, 5\u2032-CCAAGAAACAAGCGUAAUA-3\u2032; RAD18-6, 5\u2032-GGAGCAGGUUAAUGGAUAA-3\u2032; luciferase, 5\u2032-CUUACGCUGAGUACUUCGA-3\u2032 (Elbashir et al., 2001); FANCD2, 5\u2032-GGUCAGAGCUGUAUUAUUC-3\u2032 (Wagner and Karnitz, >>2009<<); PCNA, 5\u2032-GGAGAAAGUUUCAGACUAU-3\u2032 (Merkerova et al., 2007); and FANCL, 5\u2032-GACAAGAGCUGUAUGCACU-3\u2032 (Meetei et al., 2003)." . _:b444951982 . _:b444952081 . _:b444952082 . _:b444952083 . _:b444952092 . _:b444952093 . _:b444951977 . _:b444952071 . _:b444952094 . _:b444952006 . _:b444952095 . . _:b444952088 . _:b444952089 . _:b444951976 . _:b444952090 . _:b444952091 . _:b444952068 . _:b444952069 . _:b444951979 . _:b444952070 . _:b444952071 . _:b444952064 . _:b444952065 . _:b444951978 . _:b444952066 . _:b444952067 . _:b444952088 . _:b444952175 . _:b444952076 . _:b444952077 . _:b444951989 . _:b444952078 . . _:b444952079 . _:b444952072 . _:b444952073 . _:b444951988 . . _:b444952074 . _:b444951919 . _:b444952075 . _:b444952052 . _:b444952053 . _:b444951991 . _:b444952054 . . _:b444952055 . _:b444952048 . _:b444952049 . _:b8944690 . _:b444951990 . _:b444952050 . . _:b444952051 . _:b444952060 . _:b444951985 . _:b444952061 . _:b444952062 . _:b444952063 . _:b444952056 . _:b444951984 . _:b444952057 . _:b444951966 . _:b444952058 . _:b444952059 . _:b444952036 . _:b444951987 . _:b444952037 . _:b444952121 . _:b444952038 . _:b444952039 . _:b444952181 . _:b444952032 . _:b444951986 . _:b444952033 . . _:b444952034 . _:b444952035 . _:b444952044 . _:b444951997 . _:b444952045 . _:b444952046 . _:b444952047 . _:b444952040 . _:b444951996 . _:b444952041 . _:b444952047 . . _:b444952042 . _:b444952043 . _:b444952020 . _:b444951999 . _:b444952021 . _:b444952022 . _:b444952023 . _:b444952016 . _:b444951998 . _:b444952017 . _:b444952143 . _:b444952018 . _:b444952019 . _:b444951967 . _:b8944661 "Genetic data demonstrate that RAD18 and PCNA ubiquitylation also play critical roles in cells challenged with agents that induce ICLs (Yamashita et al., 2002; Tateishi et al., >>2003<<; Nojima et al., 2005; Arakawa et al., 2006; Shen et al., 2006; Simpson et al., 2006; Saberi et al., 2007)." . _:b444952028 . _:b444951993 . _:b444952029 . _:b444952030 . _:b444952221 . _:b444952031 . _:b444952024 . _:b444951992 . _:b444952025 . _:b444952026 . _:b444952027 . _:b444952004 . _:b444951995 . _:b444952005 . _:b444952011 . _:b444952006 . _:b444952099 . _:b444952007 . . _:b8944699 "We therefore asked whether PCNA ubiquitination is required for cisplatin-induced FANCD2 monoubiquitylation by modifying a previously described approach (Niimi et al., >>2008<<). Cells were transfected with an siRNA that depletes endogenous PCNA along with plasmids that express siRNA-resistant wild-type PCNA (PCNAWT) or PCNA mutated at the ubiquitin acceptor site Lys164 (PCNAK164R). The depletion of endogenous" . _:b444952000 . _:b444952010 . _:b444952001 . . _:b444951994 . _:b444952002 . _:b444952003 . _:b444952012 . _:b444952005 . _:b444952013 . . _:b444952014 . _:b444952015 . _:b444952008 . _:b444952004 . _:b444952009 . _:b444952010 . _:b444952011 . _:b444951988 . _:b444951989 . . _:b444952007 . _:b444951990 . . _:b444951991 . _:b444951984 . _:b444951985 . _:b444952006 . _:b444951986 . _:b8944690 "Previous studies demonstrated that both RAD18 and FANCD2 play important roles in eukaryotic cells exposed to cisplatin and other DNA cross-linking agents (Yamashita et al., 2002; Nojima et al., 2005; Ross et al., >>2005<<; Lee and Myung, 2008; Wagner and Karnitz, 2009; Hicks et al., 2010)." . _:b444951987 . _:b444951996 . _:b444951997 . _:b444952002 . _:b444952001 . _:b444951998 . _:b444951999 . _:b444951992 . _:b444951993 . _:b444952000 . _:b444951994 . . _:b444951995 . _:b444951972 . _:b444951973 . _:b444951989 . _:b444952003 . _:b444951974 . _:b444951975 . _:b444951968 . _:b444951969 . _:b444952002 . _:b444951970 . _:b444951971 . _:b444951980 . _:b444951981 . _:b444952013 . _:b444951982 . _:b444951954 . _:b444951983 . _:b444951976 . _:b444952067 . _:b444951977 . _:b444952012 . _:b444951978 . . _:b444951979 . _:b444951956 . _:b444951957 . _:b444952015 . _:b444951958 . _:b444951959 . . _:b444951952 . _:b444951953 . _:b444952014 . _:b444951954 . _:b444951955 . _:b444951964 . _:b444951965 . _:b444952009 . _:b444951966 . _:b444951967 . _:b444951960 . _:b444952206 . _:b444951961 . _:b444952008 . _:b444951962 . _:b444951963 . _:b444951940 . _:b444951941 . . _:b444952011 . _:b444951942 . _:b444951943 . _:b444951994 . _:b444951936 . _:b444951937 . _:b444952010 . _:b444951938 . . _:b444952076 . . _:b444951939 . _:b8944660 "Genetic data demonstrate that RAD18 and PCNA ubiquitylation also play critical roles in cells challenged with agents that induce ICLs (Yamashita et al., >>2002<<; Tateishi et al., 2003; Nojima et al., 2005; Arakawa et al., 2006; Shen et al., 2006; Simpson et al., 2006; Saberi et al., 2007)." . _:b444951948 . _:b444951949 . _:b444952021 . _:b444951950 . _:b444952003 . _:b444951951 . . _:b444951944 . _:b444951945 . _:b444952020 . _:b444951946 . . _:b444951947 . . _:b444951924 . _:b444951925 . _:b444952023 . _:b444951926 . _:b444951927 . _:b444951920 . _:b444951921 . _:b444952022 . _:b444951922 . _:b444951923 . _:b444951932 . _:b444951933 . _:b444952017 . _:b444951934 . . _:b444951935 . _:b444951928 . _:b444952166 . _:b444952015 . _:b444951929 . _:b444952016 . _:b444951930 . _:b444951931 . _:b444952019 . . _:b444952018 . _:b444952164 . _:b444951917 . _:b444952030 . _:b444952029 . _:b444951918 . _:b444951919 . _:b444952028 . _:b444952055 . . _:b444952031 . _:b444952030 . _:b444952025 . _:b444952032 . _:b444952024 . _:b444952048 . . _:b444952027 . . _:b444952026 . . _:b444952037 . _:b444952036 . _:b8944701 . _:b444952039 . _:b444952038 . . . _:b444952033 . . _:b444952069 . _:b444952032 . . _:b444952035 . . _:b444952034 . _:b8944695 "S1 B), which creates bulky DNA lesions that are bypassed by TLS polymerases after RAD18-mediated PCNA ubiquitylation (Tateishi et al., >>2000<<; Kannouche et al., 2004; Watanabe et al., 2004)." . . _:b444952045 . _:b444952044 . _:b8944693 . . _:b8944684 "The biochemical fractionation of soluble and chromatin-bound proteins was performed as described previously (Geng et al., >>2007<<). For immunoprecipitations, cells were lysed in 20 mM Tris, pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1% Triton X-100 supplemented with 5 mM CaCl2, 300 U/ml micrococcal nuclease, 10 mM N-ethylmaleimide, 10 \u00B5g/ml leupeptin, 5 \u00B5g/ml pepstatin, 20 nM" . _:b444952047 . _:b444952046 . _:b444951982 . _:b444952041 . . _:b444952040 . _:b444952043 . _:b8944656 "Ubiquitylation of the ID complex targets it to the stalled replication fork or site of damage, where it promotes processing and repair of the ICL lesion (R\u00E4schle et al., 2008; Knipscheer et al., 2009; Kratz et al., >>2010<<; Smogorzewska et al., 2010)." . . _:b444952042 . _:b444952186 . _:b8944660 . _:b444952053 . . _:b444952052 . _:b444952033 . _:b444951990 . _:b444952055 . _:b444952051 . _:b444952054 . _:b444952124 . _:b444952049 . . . _:b444952048 . _:b444952215 . _:b444952092 . _:b444952051 . . . _:b444952050 . . _:b444952061 . . _:b444952060 . _:b444951918 "10"^^ . . _:b444952063 . _:b444951919 "9"^^ . _:b444951917 "10"^^ . _:b8944659 "a known role in the recruitment and regulation of TLS polymerases, which, because of large active sites, have reduced fidelity but increased ability to synthesize over lesions that stall the high fidelity DNA polymerases (Guo et al., >>2009<<). Genetic data demonstrate that RAD18 and PCNA ubiquitylation also play critical roles in cells challenged with agents that induce ICLs (Yamashita et al., 2002; Tateishi et al., 2003; Nojima et al., 2005; Arakawa et al., 2006; Shen et al." . _:b444952062 . _:b444951988 . . _:b8944652 . _:b444952057 . _:b444952179 . _:b444952056 . _:b444952219 . . _:b444952059 . . _:b444952058 . _:b444951926 "6"^^ . . _:b444951927 "6"^^ . . _:b444952203 . _:b444951924 "6"^^ . . _:b444951977 . _:b444951925 "6"^^ . _:b444951922 "6"^^ . _:b444952045 . . _:b444951923 "6"^^ . . _:b444951920 "9"^^ . _:b444951921 "7"^^ . . _:b8944650 "introduction" . _:b444952105 . _:b444951917 . _:b444951933 "5"^^ . _:b444952118 . _:b8944664 "data demonstrate that RAD18 and PCNA ubiquitylation also play critical roles in cells challenged with agents that induce ICLs (Yamashita et al., 2002; Tateishi et al., 2003; Nojima et al., 2005; Arakawa et al., 2006; Shen et al., >>2006<<; Simpson et al., 2006; Saberi et al., 2007). However, these roles remain poorly understood." . _:b444951932 "5"^^ . _:b444951935 "5"^^ . . _:b8944688 . _:b444951934 "5"^^ . _:b444951959 . _:b444951929 "5"^^ . _:b444951930 . _:b444951928 "6"^^ . _:b444951931 "5"^^ . _:b444951930 "5"^^ . . _:b444951941 "4"^^ . _:b8944675 "5\u2032-GCUCUCUGAUCGUGAUUUA-3\u2032; RAD18-3, 5\u2032-GCAGUUUGCUUUAGAGUCA-3\u2032; RAD18-4, 5\u2032-AUAACCGCAUAUUAGAUGA-3\u2032; RAD18-5, 5\u2032-CCAAGAAACAAGCGUAAUA-3\u2032; RAD18-6, 5\u2032-GGAGCAGGUUAAUGGAUAA-3\u2032; luciferase, 5\u2032-CUUACGCUGAGUACUUCGA-3\u2032 (Elbashir et al., >>2001<<); FANCD2, 5\u2032-GGUCAGAGCUGUAUUAUUC-3\u2032 (Wagner and Karnitz, 2009); PCNA, 5\u2032-GGAGAAAGUUUCAGACUAU-3\u2032 (Merkerova et al., 2007); and FANCL, 5\u2032-GACAAGAGCUGUAUGCACU-3\u2032 (Meetei et al., 2003)." . _:b444951940 "4"^^ . . _:b444951943 "4"^^ . . . . _:b444951942 "4"^^ . _:b444951937 "5"^^ . _:b444951936 "5"^^ . _:b444952042 . _:b8944695 . _:b444952074 . _:b444951939 "4"^^ . _:b444951938 "4"^^ . _:b444951949 "4"^^ . _:b444951963 . . _:b444952139 . _:b444951948 "4"^^ . . _:b444951928 . _:b444951951 "4"^^ . _:b444952222 . _:b444952117 . _:b444951950 "4"^^ . _:b444952182 . _:b444951945 "4"^^ . _:b444952112 . _:b444952202 . . _:b444951944 "4"^^ . _:b444951947 "4"^^ . _:b444952136 . _:b444952122 . _:b444951946 "4"^^ . _:b444951950 . _:b444951957 "4"^^ . _:b8944696 . _:b444951922 . . _:b444952050 . _:b444951956 "4"^^ . _:b444951952 . _:b444951959 "3"^^ . _:b444951958 "4"^^ . _:b444952176 . . . _:b444951953 "4"^^ . _:b444952009 . _:b444952068 . _:b444951952 "4"^^ . _:b444951938 . . _:b444951955 "4"^^ . _:b8944700 . _:b444951954 "4"^^ . _:b444951981 . _:b444951965 "3"^^ . . _:b444952196 . _:b444951944 . _:b444951964 "3"^^ . _:b444951967 "3"^^ . _:b8944703 "These observations, coupled with the fact that FANCL is recruited to chromatin after DNA damage (Matsushita et al., 2005; Alpi et al., >>2008<<), prompted us to ask whether PCNA ubiquitylation affected the binding of FANCL to chromatin." . _:b444951966 "3"^^ . _:b8944708 "and REV3 (Sarkar et al., 2006; Shen et al., 2006), these results, taken in conjunction with the recent finding that FANCD2 has a PCNA interaction motif that is required for DNA damage\u2013induced FANCD2 monoubiquitylation (Howlett et al., >>2009<<), demonstrate that PCNA also serves as a central hub for the steps leading to the activation of the FA pathway in human cells." . . _:b444951961 "3"^^ . _:b8944678 . _:b444951960 "3"^^ . . _:b444951923 . _:b444951963 "3"^^ . . _:b444952130 . _:b444951962 "3"^^ . . _:b444951973 "3"^^ . _:b444951920 . _:b444951972 "3"^^ . . _:b444952226 . _:b444952188 . _:b8944696 "S1 B), which creates bulky DNA lesions that are bypassed by TLS polymerases after RAD18-mediated PCNA ubiquitylation (Tateishi et al., 2000; Kannouche et al., >>2004<<; Watanabe et al., 2004)." . _:b444951975 "3"^^ . _:b444951974 "3"^^ . . _:b444951969 "3"^^ . . . . _:b444951968 "3"^^ . _:b444951971 "3"^^ . _:b444952111 . . _:b444951970 "3"^^ . _:b444952129 . _:b444951964 . _:b444951981 "3"^^ . _:b444952107 . _:b444951980 "3"^^ . _:b444951983 "3"^^ . _:b444951982 "3"^^ . _:b444951977 "3"^^ . . _:b8944689 "Previous studies demonstrated that both RAD18 and FANCD2 play important roles in eukaryotic cells exposed to cisplatin and other DNA cross-linking agents (Yamashita et al., 2002; Nojima et al., >>2005<<; Ross et al., 2005; Lee and Myung, 2008; Wagner and Karnitz, 2009; Hicks et al., 2010)." . "10.1083%2Fjcb.201005101" . _:b444952066 . . _:b444951976 "3"^^ . _:b444951979 "3"^^ . _:b444951978 "3"^^ . _:b444952073 . _:b444951989 "3"^^ . _:b444952225 . _:b444952058 . _:b444951988 "3"^^ . _:b8944708 . _:b444951991 "3"^^ . _:b444951990 "3"^^ . . . _:b444952056 . . _:b444951985 "3"^^ . . . _:b444951984 "3"^^ . _:b444952026 . _:b444952210 . _:b444952083 . _:b444951970 . _:b444951987 "3"^^ . _:b444952165 . _:b444952163 . _:b444951986 "3"^^ . _:b444952075 . _:b444951997 "3"^^ . . _:b444951996 "3"^^ . . _:b444951999 "3"^^ . _:b444952174 . _:b444951998 "3"^^ . _:b444951940 . _:b444952147 . _:b444951993 "3"^^ . _:b444951999 . _:b444951992 "3"^^ . . _:b444951995 "3"^^ . _:b444951994 "3"^^ . _:b444951939 . . _:b444952005 "2"^^ . _:b444952004 "2"^^ . _:b444952007 "2"^^ . _:b444952006 "2"^^ . . _:b444952001 "3"^^ . _:b444952000 "3"^^ . _:b8944688 "Previous studies demonstrated that both RAD18 and FANCD2 play important roles in eukaryotic cells exposed to cisplatin and other DNA cross-linking agents (Yamashita et al., >>2002<<; Nojima et al., 2005; Ross et al., 2005; Lee and Myung, 2008; Wagner and Karnitz, 2009; Hicks et al., 2010)." . . . _:b444952003 "2"^^ . _:b444952198 . _:b444951921 . _:b444952002 "3"^^ . _:b444952013 "2"^^ . _:b444951986 . _:b444952012 "2"^^ . _:b444952152 . _:b444952015 "2"^^ . _:b444952014 "2"^^ . _:b444952024 . _:b444952009 "2"^^ . _:b444952008 "2"^^ . _:b444952011 "2"^^ . . _:b444952101 . _:b444952010 "2"^^ . _:b8944655 "Ubiquitylation of the ID complex targets it to the stalled replication fork or site of damage, where it promotes processing and repair of the ICL lesion (R\u00E4schle et al., 2008; Knipscheer et al., >>2009<<; Kratz et al., 2010; Smogorzewska et al., 2010)." . _:b444952021 "2"^^ . . _:b444952020 "2"^^ . _:b444952023 "2"^^ . _:b444952127 . _:b8944675 . _:b444952022 "2"^^ . _:b444952140 . _:b444952017 "2"^^ . _:b444952114 . _:b444952016 "2"^^ . . . _:b444952154 . _:b444952019 "2"^^ . _:b8944659 . _:b444952069 . _:b444952018 "2"^^ . _:b444952068 . _:b444952029 "2"^^ . . _:b444952097 . _:b444952071 . _:b444952028 "2"^^ . _:b444951978 . _:b444952070 . _:b444952031 "2"^^ . _:b444952065 . _:b444952134 . _:b444952030 "2"^^ . _:b444951975 . _:b444952014 . _:b444952064 . _:b444952025 "2"^^ . . _:b444952067 . _:b444952024 "2"^^ . _:b8944694 . _:b444952066 . _:b444952217 . _:b444952027 "2"^^ . _:b444952116 . _:b444952077 . _:b444952035 . _:b444952026 "2"^^ . . . _:b444952076 . _:b444952037 "2"^^ . _:b444952064 . _:b444952079 . _:b444952036 "2"^^ . _:b444952078 . . . _:b444952039 "2"^^ . _:b444952073 . . _:b444952038 "2"^^ . _:b8944652 "In response to ICLs and replication stress, FANCM and FAAP24 are recruited to replication forks stalled either by ICLs or other forms of replication stress (Ciccia et al., >>2007<<; Kim et al., 2008)." . _:b444952072 . _:b444951956 . _:b444952086 . _:b444952033 "2"^^ . _:b8944653 "In response to ICLs and replication stress, FANCM and FAAP24 are recruited to replication forks stalled either by ICLs or other forms of replication stress (Ciccia et al., 2007; Kim et al., >>2008<<). The FANCM\u2013FAAP24 complex promotes chromatin loading of the FA core complex (FANCA, FANCB, FANCC, FANCE, FANCF, FANCG, FANCL, and FAAP100), which contains FA complementation group L (FANCL) protein, an E3 ubiquitin ligase. FANCL and" . . _:b444952075 . _:b444952032 "2"^^ . . _:b444952074 . . _:b444952035 "2"^^ . _:b444952085 . _:b444952034 "2"^^ . _:b444952084 . _:b444952028 . . _:b444952091 . _:b444952045 "2"^^ . _:b444952087 . _:b444952044 "2"^^ . _:b444952194 . _:b444952086 . . . _:b444952047 "2"^^ . _:b444952081 . _:b444952046 "2"^^ . _:b444952137 . . _:b444952080 . _:b8944652 . _:b444952041 "2"^^ . . _:b444952083 . _:b8944653 . _:b444952040 "2"^^ . _:b444952082 . _:b8944654 . _:b444952043 "2"^^ . _:b444951946 . _:b444952093 . _:b8944655 . _:b444952042 "2"^^ . "PMC0" . _:b444952092 . _:b8944706 "Although previous studies suggest that ubiquitylated PCNA plays an important role in regulating the TLS polymerases REV1 and REV3 (Sarkar et al., >>2006<<; Shen et al., 2006), these results, taken in conjunction with the recent finding that FANCD2 has a PCNA interaction motif that is required for DNA damage\u2013induced FANCD2 monoubiquitylation (Howlett et al., 2009), demonstrate that PCNA also" . _:b444952053 "2"^^ . . _:b444952095 . _:b444952052 "2"^^ . _:b444952012 . _:b444952094 . . _:b444952055 "2"^^ . _:b8944650 . _:b444952089 . . _:b8944651 . _:b444952054 "2"^^ . _:b444952088 . _:b444952061 . _:b8944660 . . _:b444952049 "2"^^ . _:b444952091 . _:b8944661 . _:b8944665 . _:b8944663 . _:b444952048 "2"^^ . . _:b444952090 . _:b8944662 . _:b444952051 "2"^^ . _:b444952008 . _:b444952101 . _:b8944663 . _:b444952050 "2"^^ . _:b444952100 . _:b8944656 . _:b444952061 "2"^^ . _:b8944670 . _:b444952103 . _:b8944703 . _:b8944657 . _:b444952060 "2"^^ . _:b8944665 "that RAD18 and PCNA ubiquitylation also play critical roles in cells challenged with agents that induce ICLs (Yamashita et al., 2002; Tateishi et al., 2003; Nojima et al., 2005; Arakawa et al., 2006; Shen et al., 2006; Simpson et al., >>2006<<; Saberi et al., 2007). However, these roles remain poorly understood." . _:b8944671 . _:b444952102 . . _:b8944658 . _:b444952063 "2"^^ . . _:b8944674 . _:b444952097 . _:b8944659 . _:b444952062 "2"^^ . . _:b444952096 . _:b8944668 . _:b444952057 "2"^^ . _:b8944683 "The plasmid pET25b-hRAD18-His6hHR6B, which was used to express the RAD18\u2013RAD6B complex in E. coli, has been described previously (Notenboom et al., >>2007<<) and was provided by T." . _:b444952099 . _:b444952056 "2"^^ . _:b8944669 . . _:b444952098 . _:b8944672 "Cell cycle analyses (Arlander et al., 2003) and clonogenic assays (Wagner and Karnitz, >>2009<<) were performed as described previously." . _:b8944670 . _:b444952059 "2"^^ . _:b444952109 . _:b8944671 . _:b444952058 "2"^^ . _:b444952108 . _:b8944664 . . _:b444952111 . _:b8944665 . _:b8944670 "HeLa and K562 cells obtained from American Type Culture Collection were grown and transfected by electroporation as described previously (Arlander et al., >>2003<<) and were used 48 or 72 h after transfection as indicated. Cell cycle analyses (Arlander et al., 2003) and clonogenic assays (Wagner and Karnitz, 2009) were performed as described previously." . _:b444952110 . _:b8944666 . _:b8944682 . . _:b444952105 . _:b8944667 . _:b444952141 . _:b444952104 . _:b8944676 . _:b444952128 . _:b8944680 . _:b444952109 . _:b444952107 . _:b444952021 . _:b8944677 . . _:b444952106 . _:b444951943 . _:b8944678 . . _:b444952019 . _:b444952117 . _:b8944679 . _:b444952116 . _:b8944672 . _:b444952167 . _:b444952119 . _:b8944673 . _:b444952118 . _:b8944674 . _:b444952113 . . _:b8944675 . _:b444952104 . _:b444952112 . _:b8944684 . _:b444952115 . _:b8944685 . _:b444951924 . _:b8944661 . _:b444952114 . _:b8944686 . _:b444952125 . . . . _:b8944687 . _:b444952124 . . _:b8944680 . _:b444952220 . _:b444952080 . _:b444952029 . _:b444952127 . _:b8944681 . _:b444952216 . _:b444952126 . . _:b8944682 . _:b8944691 "Previous studies demonstrated that both RAD18 and FANCD2 play important roles in eukaryotic cells exposed to cisplatin and other DNA cross-linking agents (Yamashita et al., 2002; Nojima et al., 2005; Ross et al., 2005; Lee and Myung, >>2008<<; Wagner and Karnitz, 2009; Hicks et al., 2010)." . _:b444952121 . . _:b8944683 . _:b444952120 . _:b8944692 . _:b444952123 . _:b8944693 . . _:b444952122 . _:b8944694 . _:b444952077 . . _:b444952133 . _:b8944695 . _:b444952132 . . _:b8944688 . _:b444952135 . _:b444951983 . _:b8944689 . _:b8944664 . _:b444952134 . _:b8944690 . _:b444952129 . _:b444952209 . _:b8944691 . . _:b444952128 . . _:b8944700 . _:b444952041 . _:b444952131 . _:b444952185 . _:b8944705 . _:b8944701 . _:b8944668 . _:b444952130 . _:b8944702 . _:b444951955 . . _:b444952141 . . _:b8944703 . . _:b444952192 . _:b444952140 . _:b444952025 . _:b8944696 . _:b444952143 . _:b8944697 . _:b444952142 . _:b8944698 . _:b444952137 . _:b8944699 . . _:b444952136 . . _:b8944708 . _:b444952013 . . _:b444952139 . _:b444951927 . . _:b444952138 . . _:b444952149 . . _:b444952148 . _:b8944704 . . _:b444952151 . _:b8944705 . _:b8944667 "One function for ubiquitylated PCNA in the repair of ICLs is to recruit or regulate the TLS DNA polymerases REV1 and polymerase \u03B6, which are required for ICL repair (Sarkar et al., >>2006<<; Shen et al., 2006)." . _:b444951996 . _:b444952150 . _:b444952126 . _:b8944706 . . _:b444952145 . _:b8944707 . _:b8944698 "S1 D) and reduced the cisplatin-induced binding of FANCD2 to chromatin, an event that depends on FANCD2 ubiquitylation (Wang et al., >>2004<<), in K562 (Fig." . _:b444952144 . . . _:b444952147 . _:b8944689 . . _:b444952146 . _:b444952157 . . _:b444952156 . . _:b444952159 . _:b8944701 "However, a very recent crystallographic structural analysis of Drosophila melanogaster FANCL has further refined our understanding of FANCL\u2019s structure (Cole et al., >>2010<<). This study showed that the N terminus contains two separable domains, an E2-like fold (ELF) domain and a DRWD domain. To test which FANCL domain is important for PCNA binding, we examined binding to the entire N-terminal fragment (which" . _:b444952158 . _:b444952153 . _:b444952152 . _:b444952155 . . . _:b444951992 . _:b444952154 . _:b444951934 . _:b444952095 . _:b444952165 . _:b444952155 . _:b444951991 . _:b444952164 . _:b444952167 . _:b444952084 . . . _:b444952166 . . _:b444952161 . _:b444952027 . . _:b444952160 . . _:b444952163 . _:b444952162 . _:b444952173 . . _:b444952172 . _:b444952175 . _:b444952174 . _:b8944651 . _:b444952212 . . _:b444952169 . . _:b444952157 . _:b444952168 . . _:b444952171 . _:b444952144 . _:b8944666 "also play critical roles in cells challenged with agents that induce ICLs (Yamashita et al., 2002; Tateishi et al., 2003; Nojima et al., 2005; Arakawa et al., 2006; Shen et al., 2006; Simpson et al., 2006; Saberi et al., >>2007<<). However, these roles remain poorly understood." . _:b444952170 . . _:b444952190 . _:b444952181 . . . _:b444952180 . . _:b444951998 . _:b444952183 . . _:b444952183 . . _:b444952182 . _:b444951985 . _:b444952177 . . . _:b444952176 . _:b8944662 . . . _:b444952179 . _:b444952178 . _:b8944692 . . . _:b8944702 "These observations, coupled with the fact that FANCL is recruited to chromatin after DNA damage (Matsushita et al., >>2005<<; Alpi et al., 2008), prompted us to ask whether PCNA ubiquitylation affected the binding of FANCL to chromatin." . _:b444952189 . _:b444951935 . _:b444952059 . _:b444952188 . _:b444952191 . . _:b444952190 . . _:b444952185 . . _:b444952184 . _:b8944707 . . _:b444952187 . _:b444952186 . . _:b444952197 . _:b444952094 . _:b444952196 . _:b444952208 . . _:b444952199 . _:b444951980 . _:b444952198 . . _:b444952149 . . _:b444952193 . _:b444952187 . . _:b444951937 . _:b444952192 . _:b444952195 . _:b444952194 . _:b8944676 . _:b444952052 . _:b444952205 . . . . _:b444952204 . _:b444952207 . _:b444951968 . _:b8944672 . _:b444952206 . _:b444952201 . _:b444952082 . _:b444952200 . _:b444952203 . _:b444952195 . _:b444952202 . . _:b8944683 . . _:b444952213 . _:b444952212 . . . _:b444952215 . _:b8944686 . . . _:b444952214 . . . _:b444952209 . _:b444952036 . _:b444952208 . _:b8944680 "The FANCL expression plasmids were created by ligating PCR-amplified FANCL into vectors that append an N-terminal SFB tag (pSFB; Chini and Chen, 2006), an N-terminal S tag (pSPN; Hackbarth et al., >>2004<<), or a C-terminal tandem HA tag (pcDNA3-HA2; Volkmer and Karnitz, 1999). The S-PCNA expression vector was constructed by ligating PCR-amplified PCNA into pSPC, which appends a C-terminal S tag (Hackbarth et al., 2004)." . _:b444952211 . . _:b444952210 . _:b444952221 . . _:b444952220 . . _:b444952223 . . _:b444952222 . . _:b444952217 . . _:b444952216 . . _:b444952219 . . _:b444952218 . _:b444952000 . . . . _:b444952228 . . . _:b444952218 . _:b8944668 "One function for ubiquitylated PCNA in the repair of ICLs is to recruit or regulate the TLS DNA polymerases REV1 and polymerase \u03B6, which are required for ICL repair (Sarkar et al., 2006; Shen et al., >>2006<<). Nevertheless, PCNA ubiquitylation may have a previously unknown function in the regulation of the FA pathway. Consistent with this later possibility, we report in this study that RAD18-mediated PCNA ubiquitylation plays a pivotal role" . _:b8944693 "and FANCD2 play important roles in eukaryotic cells exposed to cisplatin and other DNA cross-linking agents (Yamashita et al., 2002; Nojima et al., 2005; Ross et al., 2005; Lee and Myung, 2008; Wagner and Karnitz, 2009; Hicks et al., >>2010<<). In our experiments to understand the DNA repair pathways that affect the sensitivity of human tumor cells to cisplatin, a chemotherapy agent that causes ICL formation (Wang and Lippard, 2005), we depleted HeLa cells of FANCD2, a key" . _:b8944667 . _:b444952016 . _:b444952225 . _:b8944681 . _:b444952224 . . _:b444952227 . _:b444952172 . _:b444952226 . . . . . _:b8944677 "RAD18-6, 5\u2032-GGAGCAGGUUAAUGGAUAA-3\u2032; luciferase, 5\u2032-CUUACGCUGAGUACUUCGA-3\u2032 (Elbashir et al., 2001); FANCD2, 5\u2032-GGUCAGAGCUGUAUUAUUC-3\u2032 (Wagner and Karnitz, 2009); PCNA, 5\u2032-GGAGAAAGUUUCAGACUAU-3\u2032 (Merkerova et al., >>2007<<); and FANCL, 5\u2032-GACAAGAGCUGUAUGCACU-3\u2032 (Meetei et al., 2003)." . _:b444952115 . _:b444952098 . _:b8944687 "results and discussion" . _:b444952090 . . _:b444952081 . _:b444951936 . _:b444952093 . _:b8944651 "To date, 13 FA genetic complementation groups have been identified (Moldovan and D\u2019Andrea, >>2009<<). Consistent with the sensitivity to ICLs, the proteins encoded by the FANC genes repair these lesions. Although the molecular and biochemical functions of the FA pathway remain poorly understood, the following model has emerged. In" . . . . . _:b444952177 . _:b444951948 . _:b444952022 . _:b444952119 . _:b444952170 . . _:b444951942 . _:b444952191 . _:b444952063 . _:b8944685 . . _:b8944654 "Ubiquitylation of the ID complex targets it to the stalled replication fork or site of damage, where it promotes processing and repair of the ICL lesion (R\u00E4schle et al., >>2008<<; Knipscheer et al., 2009; Kratz et al., 2010; Smogorzewska et al., 2010)." . . _:b8944700 "domains: the N terminus (amino acids 1\u2013306) was predicted to contain three WD40 domains, and the C terminus (amino acids 307\u2013375) was predicted to contain a zinc-binding region that is essential for E3 ligase activity (Meetei et al., >>2003<<). However, a very recent crystallographic structural analysis of Drosophila melanogaster FANCL has further refined our understanding of FANCL\u2019s structure (Cole et al., 2010)." . . _:b444952199 . . . _:b444951929 . _:b444952043 . . . . . . _:b444952110 . . . _:b444951958 . . . _:b444952189 . . _:b8944657 . . _:b444952085 . . . _:b444951953 . . _:b8944707 "Although previous studies suggest that ubiquitylated PCNA plays an important role in regulating the TLS polymerases REV1 and REV3 (Sarkar et al., 2006; Shen et al., >>2006<<), these results, taken in conjunction with the recent finding that FANCD2 has a PCNA interaction motif that is required for DNA damage\u2013induced FANCD2 monoubiquitylation (Howlett et al., 2009), demonstrate that PCNA also serves as a" . . _:b444952138 . . _:b444952070 . _:b8944679 . _:b444951925 . _:b8944687 _:b8944704 . _:b8944687 _:b8944705 . _:b8944687 _:b8944706 . _:b8944687 _:b8944707 . _:b8944687 _:b8944708 . _:b8944656 . _:b444952072 . . . _:b444951993 . _:b444952135 . _:b8944706 . _:b444951941 . _:b8944681 "created by ligating PCR-amplified FANCL into vectors that append an N-terminal SFB tag (pSFB; Chini and Chen, 2006), an N-terminal S tag (pSPN; Hackbarth et al., 2004), or a C-terminal tandem HA tag (pcDNA3-HA2; Volkmer and Karnitz, >>1999<<). The S-PCNA expression vector was constructed by ligating PCR-amplified PCNA into pSPC, which appends a C-terminal S tag (Hackbarth et al., 2004)." . . . _:b444952023 . . _:b444952168 . _:b444952017 . _:b444952200 . _:b444952103 . . _:b444951951 . _:b8944705 "However, it is worth noting that PCNA is still ubiquitylated in RAD18\u2212/\u2212 cells and that cells expressing PCNAK164R are more sensitive to cisplatin than are RAD18\u2212/\u2212 cells (Simpson et al., >>2006<<), thus raising the possibility that the lack of epistasis could be explained by the residual PCNA ubiquitylation in RAD18\u2212/\u2212 cells." . . _:b444952178 . _:b8944687 _:b8944696 . _:b8944687 _:b8944697 . _:b8944687 _:b8944698 . _:b8944686 "To prepare in vitro\u2013ubiquitylated PCNA, 100 ng purified GST-PCNA or GST-PCNAK164R was incubated with 50 ng rabbit E1 ubiquitin\u2013activating enzyme (EMD) and 350 ng hRAD18-RAD6 (prepared as described previously in Notenboom et al. [>>2007<<]) with or without 5 \u00B5g HA-tagged human ubiquitin (Boston Biochem) in ubiquitination reaction buffer (50 mM Tris, pH 7.4, 5 mM MgCl2, 2 mM ATP, 2 \u00B5M ZnCl2, 2 mM NaF, and 0.6 mM DTT) at 30\u00B0C for 1 h." . _:b8944687 _:b8944699 . . _:b8944687 _:b8944700 . _:b444952120 . _:b8944687 _:b8944701 . . _:b8944687 _:b8944702 . _:b8944687 _:b8944703 . _:b8944687 _:b8944688 . . _:b8944687 _:b8944689 . _:b444951960 . _:b8944687 _:b8944690 . _:b8944687 _:b8944691 . _:b8944687 _:b8944692 . . _:b8944687 _:b8944693 . _:b8944687 _:b8944694 . _:b8944687 _:b8944695 . . . . . . . _:b8944704 "of FANCC, which encodes an FA core complex member, and RAD18 caused greater sensitivity to cisplatin than did either individual gene disruption alone, suggesting that the FA and RAD18 pathways are not epistatic (Hirano et al., >>2005<<). In contrast, our siRNA codepletion experiments suggest that the FA and RAD18 pathways may be epistatic in terms of cisplatin sensitivity (Fig." . . . . _:b8944669 _:b8944670 . _:b8944669 _:b8944671 . _:b444951971 . _:b444952180 . _:b444952069 "2"^^ . _:b444952201 . _:b444952068 "2"^^ . _:b444952071 "2"^^ . _:b444952044 . _:b444952207 . _:b444952070 "2"^^ . _:b444952065 "2"^^ . _:b444952102 . _:b444952064 "2"^^ . _:b8944669 _:b8944672 . _:b8944669 _:b8944673 . _:b444952067 "2"^^ . _:b8944669 _:b8944674 . _:b444951957 . _:b8944669 _:b8944675 . _:b8944669 _:b8944676 . _:b8944669 _:b8944677 . _:b444952066 "2"^^ . _:b8944669 _:b8944678 . _:b444952079 . _:b8944669 _:b8944679 . _:b8944669 _:b8944680 . _:b444952145 . _:b8944669 _:b8944681 . _:b444952077 "2"^^ . _:b8944669 _:b8944682 . _:b8944669 _:b8944683 . _:b8944669 _:b8944684 . _:b8944669 _:b8944685 . _:b444952076 "2"^^ . _:b8944669 _:b8944686 . _:b8944650 _:b8944652 . _:b8944650 _:b8944653 . _:b444952079 "2"^^ . _:b8944650 _:b8944654 . _:b8944650 _:b8944655 . _:b444951918 . . . _:b444952087 . _:b444952078 "2"^^ . _:b444952142 . _:b8944650 _:b8944651 . _:b444952073 "2"^^ . _:b444952227 . _:b444952072 "2"^^ . _:b8944650 _:b8944668 . . _:b444952075 "2"^^ . _:b8944650 _:b8944664 . . _:b8944650 _:b8944665 . _:b444952074 "2"^^ . _:b8944650 _:b8944666 . _:b8944650 _:b8944667 . _:b444951931 . _:b8944650 _:b8944660 . _:b444952151 . _:b8944650 _:b8944661 . _:b444952085 "2"^^ . _:b8944650 _:b8944662 . _:b8944650 _:b8944663 . _:b8944650 _:b8944656 . _:b8944650 _:b8944657 . _:b444952084 "2"^^ . _:b8944650 _:b8944658 . _:b8944650 _:b8944659 . _:b444952173 . _:b444952125 . . _:b444952087 "2"^^ . . _:b444952086 "2"^^ . _:b444952160 . _:b444951973 . _:b444952081 "2"^^ . _:b8944658 "ICLs and other forms of replication stress also activate the translesion synthesis (TLS) pathway (Lee and Myung, >>2008<<), which is initiated by the recruitment of the RAD18\u2013RAD6 complex, an E3 ubiquitin ligase and an E2 ubiquitin\u2013conjugating enzyme pair." . _:b444952080 "2"^^ . _:b444951961 . _:b444952211 . _:b444952083 "2"^^ . _:b8944679 "The FANCL expression plasmids were created by ligating PCR-amplified FANCL into vectors that append an N-terminal SFB tag (pSFB; Chini and Chen, >>2006<<), an N-terminal S tag (pSPN; Hackbarth et al., 2004), or a C-terminal tandem HA tag (pcDNA3-HA2; Volkmer and Karnitz, 1999)." . . _:b444952082 "2"^^ . _:b444952093 "2"^^ . . . _:b444952053 . _:b444952092 "2"^^ . _:b444952095 "2"^^ . _:b444951972 . _:b8944663 "Genetic data demonstrate that RAD18 and PCNA ubiquitylation also play critical roles in cells challenged with agents that induce ICLs (Yamashita et al., 2002; Tateishi et al., 2003; Nojima et al., 2005; Arakawa et al., >>2006<<; Shen et al., 2006; Simpson et al., 2006; Saberi et al., 2007)." . _:b444952094 "2"^^ . _:b444951974 . _:b444952148 . _:b444952089 "2"^^ . _:b8944677 . _:b444952146 . _:b444952088 "2"^^ . _:b444952091 "2"^^ . _:b444952090 "2"^^ . _:b444952060 . _:b444952101 "2"^^ . . _:b444952100 "2"^^ . _:b8944699 . _:b444952103 "2"^^ . _:b444952102 "2"^^ . . _:b444952097 "2"^^ . . _:b444952096 "2"^^ . _:b444952057 . . _:b444952099 "2"^^ . _:b444952098 "2"^^ . . _:b444952109 "2"^^ . . _:b444952108 "2"^^ . _:b444951947 . . _:b444952111 "2"^^ . . . . _:b444952110 "2"^^ . _:b444951962 . _:b8944687 . _:b444952105 "2"^^ . _:b444951932 . _:b444952104 "2"^^ . _:b444952107 "2"^^ . _:b444952193 . . _:b444952106 "2"^^ . _:b444952169 . _:b444952117 "2"^^ . . _:b444952171 . _:b444952116 "2"^^ . _:b444952119 "2"^^ . _:b8944704 . _:b444952132 . _:b444952118 "2"^^ . . _:b444952113 "2"^^ . _:b444952123 . _:b8944650 . _:b444952112 "2"^^ . . . _:b444952115 "2"^^ . _:b444952114 "2"^^ . _:b8944669 . _:b444952125 "2"^^ . _:b444952124 "2"^^ . _:b444952127 "2"^^ . . _:b444952126 "2"^^ . _:b444952121 "2"^^ . _:b8944653 . . _:b444952120 "2"^^ . . _:b444952123 "2"^^ . . . . _:b444952122 "2"^^ . _:b444952228 . . _:b444952133 "2"^^ . . . _:b444952132 "2"^^ . . _:b444952135 "2"^^ . _:b444952134 "2"^^ . . _:b444952129 "2"^^ . _:b444952128 "2"^^ . _:b444952131 "2"^^ . _:b444952130 "2"^^ . _:b444952089 . . . _:b444952141 "2"^^ . _:b444952205 . _:b444952140 "2"^^ . _:b8944669 "materials and methods" . _:b444951965 . _:b444952143 "2"^^ . . _:b444952142 "2"^^ . _:b444952137 "2"^^ . . _:b444952136 "2"^^ . _:b8944682 "The S-PCNA expression vector was constructed by ligating PCR-amplified PCNA into pSPC, which appends a C-terminal S tag (Hackbarth et al., >>2004<<). S-PCNA K164R was derived from pSPC-PCNA by site-directed mutagenesis. PCNA expression plasmids resistant to the siRNA, four silent mutations (identified by underlines), were generated using the oligonucleotide" . . _:b444952139 "2"^^ . _:b444952020 . . _:b444952138 "2"^^ . . _:b444952005 . _:b444952149 "2"^^ . . . _:b8944694 "In our experiments to understand the DNA repair pathways that affect the sensitivity of human tumor cells to cisplatin, a chemotherapy agent that causes ICL formation (Wang and Lippard, >>2005<<), we depleted HeLa cells of FANCD2, a key participant in the FA repair pathway, and of RAD18, a regulator of the TLS pathway (Fig." . _:b444952148 "2"^^ . _:b8944702 . _:b444952151 "2"^^ . . _:b444952150 "2"^^ . . _:b8944691 . _:b444952145 "2"^^ . _:b444952078 . _:b444952144 "2"^^ . . _:b444952147 "2"^^ . . . _:b444952146 "2"^^ . . _:b444952157 "2"^^ . _:b444951984 . _:b444952156 "2"^^ . _:b8944654 . _:b444951926 . _:b444951997 . _:b444952159 "2"^^ . _:b8944662 "Genetic data demonstrate that RAD18 and PCNA ubiquitylation also play critical roles in cells challenged with agents that induce ICLs (Yamashita et al., 2002; Tateishi et al., 2003; Nojima et al., >>2005<<; Arakawa et al., 2006; Shen et al., 2006; Simpson et al., 2006; Saberi et al., 2007)." .