title: N-terminal domains of human DNA polymerase lambda promote primer realignment during translesion DNA synthesis.
abstract: The X-family DNA polymerases λ (Polλ) and β (Polβ) possess similar 5'-2-deoxyribose-5-phosphate lyase (dRPase) and polymerase domains. Besides these domains, Polλ also possesses a BRCA1 C-terminal (BRCT) domain and a proline-rich domain at its N terminus. However, it is unclear how these non-enzymatic domains contribute to the unique biological functions of Polλ. Here, we used primer extension assays and a newly developed high-throughput short oligonucleotide sequencing assay (HT-SOSA) to compare the efficiency of lesion bypass and fidelity of human Polβ, Polλ and two N-terminal deletion constructs of Polλ during the bypass of either an abasic site or an 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) lesion. We demonstrate that the BRCT domain of Polλ enhances the efficiency of abasic site bypass by approximately 1.6-fold. In contrast, deletion of the N-terminal domains of Polλ did not affect the efficiency of 8-oxodG bypass relative to nucleotide incorporations opposite undamaged dG. HT-SOSA analysis demonstrated that Polλ and Polβ preferentially generated -1 or -2 frameshift mutations when bypassing an abasic site and the single or double base deletion frequency was highly sequence dependent. Interestingly, the BRCT and proline-rich domains of Polλ cooperatively promoted the generation of -2 frameshift mutations when the abasic site was situated within a sequence context that was susceptible to homology-driven primer realignment. Furthermore, both N-terminal domains of Polλ increased the generation of -1 frameshift mutations during 8-oxodG bypass and influenced the frequency of substitution mutations produced by Polλ opposite the 8-oxodG lesion. Overall, our data support a model wherein the BRCT and proline-rich domains of Polλ act cooperatively to promote primer/template realignment between DNA strands of limited sequence homology. This function of the N-terminal domains may facilitate the role of Polλ as a gap-filling polymerase within the non-homologous end joining pathway.
authors.affiliation: Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH, USA.
authors.forename: David J
authors.initials: DJ
authors.lastname: Taggart
authors.affiliation: Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH, USA; The Ohio State Biochemistry Program, The Ohio State University, Columbus, OH, USA.
authors.forename: Daniel M
authors.initials: DM
authors.lastname: Dayeh
authors.affiliation: Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH, USA; Department of Microbiology, The Ohio State University, Columbus, OH, USA.
authors.forename: Saul W
authors.initials: SW
authors.lastname: Fredrickson
authors.affiliation: Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH, USA; The Ohio State Biochemistry Program, The Ohio State University, Columbus, OH, USA. Electronic address: suo.3@osu.edu.
authors.forename: Zucai
authors.initials: Z
authors.lastname: Suo
mesh_terms: D002460:Cell Line; D019951:DNA Polymerase beta / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair; D016368:Frameshift Mutation; D017353:Gene Deletion; D006147:Guanine / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D055232:Proline-Rich Protein Domains
chemical_list: D017931:DNA Primers; C024829:8-hydroxyguanine; D006147:Guanine; C415367:DNA polymerase beta2; D019951:DNA Polymerase beta
keywords: Abasic site; Lesion bypass; Mutagenic analysis; Translesion DNA synthesis; X-family DNA polymerases
references.citation: Proc Natl Acad Sci U S A. 1993 Sep 1;90(17):7915-22
references.pmid: 8367443
references.citation: J Biol Chem. 2004 Jan 2;279(1):805-11
references.pmid: 14561766
references.citation: J Biol Chem. 2006 Jul 14;281(28):19038-44
references.pmid: 16675458
references.citation: J Biol Chem. 2005 Feb 4;280(5):3665-74
references.pmid: 15561706
references.citation: Bioessays. 1999 Mar;21(3):238-46
references.pmid: 10333733
references.citation: Chem Res Toxicol. 2012 Mar 19;25(3):730-40
references.pmid: 22324639
references.citation: Proc Natl Acad Sci U S A. 1990 Jun;87(12):4533-7
references.pmid: 2352934
references.citation: J Biol Chem. 1995 Jan 13;270(2):949-57
references.pmid: 7822335
references.citation: Biochemistry. 1997 Sep 16;36(37):11205-15
references.pmid: 9287163
references.citation: PLoS One. 2010 Aug 18;5(8):e12229
references.pmid: 20805875
references.citation: J Biol Chem. 2002 Dec 13;277(50):48434-40
references.pmid: 12368291
references.citation: Nucleic Acids Res. 2011 Jan;39(2):609-22
references.pmid: 20846959
references.citation: Nat Struct Mol Biol. 2009 Sep;16(9):967-72
references.pmid: 19701199
references.citation: J Mol Biol. 2007 Apr 13;367(5):1258-69
references.pmid: 17321545
references.citation: Biochem Biophys Res Commun. 2004 Oct 29;323(4):1328-33
references.pmid: 15451442
references.citation: Cell. 2006 Jan 27;124(2):331-42
references.pmid: 16439207
references.citation: Nucleic Acids Res. 2000 Sep 15;28(18):3684-93
references.pmid: 10982892
references.citation: Nucleic Acids Res. 2005 Sep 20;33(16):5354-61
references.pmid: 16174846
references.citation: Biochemistry. 1972 Sep 12;11(19):3618-23
references.pmid: 4559796
references.citation: DNA Repair (Amst). 2011 Jan 2;10(1):24-33
references.pmid: 20961817
references.citation: Biochemistry. 1972 Sep 12;11(19):3610-8
references.pmid: 4626532
references.citation: Cold Spring Harb Symp Quant Biol. 1966;31:77-84
references.pmid: 5237214
references.citation: J Biol Chem. 2005 Sep 9;280(36):31641-7
references.pmid: 16002405
references.citation: Nucleic Acids Res. 2013 Apr;41(8):e96
references.pmid: 23470999
references.citation: DNA Repair (Amst). 2010 Feb 4;9(2):109-19
references.pmid: 20006562
references.citation: J Biol Chem. 1997 Jan 24;272(4):2559-69
references.pmid: 8999973
references.citation: J Biol Chem. 2002 Apr 12;277(15):13184-91
references.pmid: 11821417
references.citation: Nucleic Acids Res. 2006 May 31;34(10):2998-3007
references.pmid: 16738138
references.citation: Nat Struct Mol Biol. 2005 Jan;12(1):97-8
references.pmid: 15608652
references.citation: Biochemistry. 2004 Jun 1;43(21):6751-62
references.pmid: 15157109
references.citation: Biochemistry. 2004 Sep 14;43(36):11605-15
references.pmid: 15350147
references.citation: Cold Spring Harb Symp Quant Biol. 2000;65:81-91
references.pmid: 12760023
references.citation: Proc Natl Acad Sci U S A. 1990 Jul;87(13):4946-50
references.pmid: 2195542
references.citation: J Biol Chem. 1988 Oct 15;263(29):14784-9
references.pmid: 3049589
references.citation: EMBO J. 1997 Jun 2;16(11):3341-8
references.pmid: 9214649
references.citation: Mol Cell. 2004 Dec 3;16(5):701-13
references.pmid: 15574326
references.citation: Carcinogenesis. 2000 Mar;21(3):361-70
references.pmid: 10688856
references.citation: Mutat Res. 2010 Nov 28;703(1):18-23
references.pmid: 20696268
references.citation: Nature. 1996 Jan 11;379(6561):183-6
references.pmid: 8538772
references.citation: DNA Repair (Amst). 2005 Dec 8;4(12):1358-67
references.pmid: 16213194
references.citation: J Biol Chem. 2002 May 24;277(21):18454-8
references.pmid: 11886860