title: Mutations in the ubiquitin binding UBZ motif of DNA polymerase eta do not impair its function in translesion synthesis during replication.
abstract: Treatment of Saccharomyces cerevisiae cells with DNA-damaging agents elicits lysine 164-linked PCNA monoubiquitination by Rad6-Rad18. Recently, a number of ubiquitin (Ub) binding domains (UBDs) have been identified in translesion synthesis (TLS) DNA polymerases and it has been proposed that the UBD in a TLS polymerase affects its binding to Ub on PCNA and that this binding mode is indispensable for a TLS polymerase to access PCNA at the site of a stalled replication fork. To evaluate the contribution of the binding of UBDs to the Ub moiety on PCNA in TLS, we have examined the effects of mutations in the C2H2 zinc binding motif and in the conserved D570 residue that lies in the alpha-helix portion of the UBZ domain of yeast Poleta. We find that mutations in the C2H2 motif have no perceptible effect on UV sensitivity or UV mutagenesis, whereas a mutation of the D570 residue adversely affects Poleta function. The stimulation of DNA synthesis by Poleta with PCNA or Ub-PCNA was not affected by mutations in the C2H2 motif or the D570 residue. These observations lead us to suggest that the binding of Ub on PCNA via its UBZ domain is not a necessary requirement for the ability of polymerase eta to function in TLS during replication.
authors: Department of Biochemistry and Molecular Biology, University of Texas Medical Branch at Galveston, 6.104 Blocker Medical Research Building, 301 University Blvd., Galveston, TX 77555-1061, USA.
authors: Narottam
authors: N
authors: Acharya
authors: Amrita
authors: A
authors: Brahma
authors: Lajos
authors: L
authors: Haracska
authors: Louise
authors: L
authors: Prakash
authors: Satya
authors: S
authors: Prakash
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000378:metabolism; D004249:DNA Damage; D004252:DNA Mutational Analysis; D004260:DNA Repair; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D025801:Ubiquitin / Q000378:metabolism*; D014466:Ultraviolet Rays; D000099270:Y-Family DNA Polymerases
chemical_list: D001224:Aspartic Acid; D004259:DNA-Directed DNA Polymerase; D018809:Proliferating Cell Nuclear Antigen; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; C112338:Rad30 protein; D000099270:Y-Family DNA Polymerases
references: EMBO J. 2000 Jul 3;19(13):3388-97
references: 10880451
references: Mol Cell. 2007 Jun 22;26(6):891-8
references: 17588522
references: Nat Struct Biol. 2001 May;8(5):417-22
references: 11323716
references: Mol Cell. 2001 Aug;8(2):407-15
references: 11545742
references: Mol Cell Biol. 2001 Nov;21(21):7199-206
references: 11585903
references: J Mol Biol. 2001 Nov 9;313(5):955-63
references: 11700052
references: Proc Natl Acad Sci U S A. 2001 Dec 4;98(25):14256-61
references: 11724965
references: Mol Cell Biol. 2002 Feb;22(3):784-91
references: 11784855
references: Mol Cell Biol. 2002 Apr;22(7):2419-26
references: 11884624
references: Nature. 2002 Sep 12;419(6903):135-41
references: 12226657
references: Nature. 2003 Sep 11;425(6954):188-91
references: 12968183
references: Mol Cell Biol. 2004 May;24(10):4267-74
references: 15121847
references: Mol Cell. 2004 May 21;14(4):491-500
references: 15149598
references: Gene. 1988 Dec 30;74(2):527-34
references: 3073106
references: Genes Dev. 1994 Apr 1;8(7):811-20
references: 7926769
references: Science. 1996 Jun 14;272(5268):1646-9
references: 8658138
references: J Biol Chem. 1997 Sep 12;272(37):23360-5
references: 9287349
references: Science. 1999 Feb 12;283(5404):1001-4
references: 9974380
references: Cell. 1999 Mar 5;96(5):645-53
references: 10089880
references: Annu Rev Biochem. 2005;74:317-53
references: 15952890
references: Science. 2005 Dec 16;310(5755):1821-4
references: 16357261
references: Proc Natl Acad Sci U S A. 2005 Dec 20;102(51):18361-6
references: 16344468
references: Proc Natl Acad Sci U S A. 2006 Apr 25;103(17):6477-82
references: 16611731
references: EMBO J. 2006 Jun 21;25(12):2847-55
references: 16763556
references: Methods Enzymol. 2006;408:390-407
references: 16793382
references: Nucleic Acids Res. 2007;35(3):881-9
references: 17251197
references: EMBO Rep. 2007 Mar;8(3):247-51
references: 17304240
references: Genes Dev. 2001 Apr 15;15(8):945-54
references: 11316789