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Molecular and Cellular Biology, October 2007, p. 7266-7272, Vol. 27, No. 20
0270-7306/07/$08.00+0     doi:10.1128/MCB.01196-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Mutations in the Ubiquitin Binding UBZ Motif of DNA Polymerase Do Not Impair Its Function in Translesion Synthesis during Replication

Narottam Acharya,¹ Amrita Brahma,¹ Lajos Haracska,² Louise Prakash,¹ and Satya Prakash^1*

Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, Texas 77555-1061,¹ Institute of Genetics, Biological Research Center, Hungarian Academy of Sciences, Szeged, Hungary²

Received 5 July 2007/ Returned for modification 23 July 2007/ Accepted 7 August 2007

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 -helix portion of the UBZ domain of yeast Pol. 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 Pol function. The stimulation of DNA synthesis by Pol 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 to function in TLS during replication.

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* Corresponding author. Mailing address: 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. Phone: (409) 747-8602. Fax: (409) 747-8608. E-mail: s.prakash{at}utmb.edu

Published ahead of print on 20 August 2007.

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Molecular and Cellular Biology, October 2007, p. 7266-7272, Vol. 27, No. 20
0270-7306/07/$08.00+0     doi:10.1128/MCB.01196-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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