Mutations in the ubiquitin binding UBZ motif of DNA polymerase eta do not impair its function in translesion synthesis during replication.
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.
Acharya
Narottam
N
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.
Brahma
Amrita
A
Haracska
Lajos
L
Prakash
Louise
L
Prakash
Satya
S
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
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
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