Structure of the Catalytic Core of S. cerevisiae DNA Polymerase ηImplications for Translesion DNA Synthesis

Structural Biology Program, Department of Physiology and Biophysics, Mount Sinai School of Medicine, New York, NY 10029, USA.
Molecular Cell (Impact Factor: 14.02). 09/2001; 8(2):417-26. DOI: 10.1016/S1097-2765(01)00306-9
Source: PubMed


DNA polymerase eta is unique among eukaryotic polymerases in its proficient ability to replicate through a variety of distorting DNA lesions. We report here the crystal structure of the catalytic core of S. cerevisiae DNA polymerase eta, determined at 2.25A resolution. The structure reveals a novel polydactyl right hand-shaped molecule with a unique polymerase-associated domain. We identify the catalytic residues and show that the fingers and thumb domains are unusually small and stubby. In particular, the unexpected absence of helices "O" and "O1" in the fingers domain suggests that openness of the active site is the critical feature which enables DNA polymerase eta to replicate through DNA lesions such as a UV-induced cis-syn thymine-thymine dimer.

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Available from: Carlos R. Escalante
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    • "Domain structures of downstream effectors of modified PCNA are indicated schematically, according to Table 1. Note that CRL4 is omitted from this figure, as the details of its interaction with PCNA have not been elucidated Structural information is available for the catalytic core of Polη, its PIP box bound to PCNA, its UBZ domain in complex with ubiquitin and a recombinant construct mimicking the ubiquitylated form of PCNA (Trincao et al. 2001; Bomar et al. 2007; Hishiki et al. 2009; Freudenthal et al. 2010). This has allowed the modelling of a complex between Polη and the ubiquitylated clamp (Freudenthal et al. 2010), in which the concept of how the polymerase acts as a reader of modified PCNA is nicely illustrated (Fig. 4a): the long, flexible C terminus of Polη, harbouring the UBZ and PIP motifs in close proximity, is able to reach over the surface of PCNA such that the two domains simultaneously contact the ubiquitin moiety and the IDCL of PCNA. "
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    ABSTRACT: The eukaryotic sliding clamp, proliferating cell nuclear antigen (PCNA), acts as a central coordinator of DNA transactions by providing a multivalent interaction surface for factors involved in DNA replication, repair, chromatin dynamics and cell cycle regulation. Posttranslational modifications (PTMs), such as mono- and polyubiquitylation, sumoylation, phosphorylation and acetylation, further expand the repertoire of PCNA’s binding partners. These modifications affect PCNA’s activity in the bypass of lesions during DNA replication, the regulation of alternative damage processing pathways such as homologous recombination and DNA interstrand cross-link repair, or impact on the stability of PCNA itself. In this review, we summarise our current knowledge about how the PTMs are “read” by downstream effector proteins that mediate the appropriate action. Given the variety of interaction partners responding to PCNA’s modified forms, the ensemble of PCNA modifications serves as an instructive model for the study of biological signalling through PTMs in general. Electronic supplementary material The online version of this article (doi:10.1007/s00412-013-0410-4) contains supplementary material, which is available to authorized users.
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    • "Translesion synthesis by these alternative polymerases is inaccurate, increasing the mutation rate in XPV cells and greatly increasing the likelihood of skin cancer in patients with XPV. The first Y-family polymerase crystal structures (Ling et al., 2001; Silvian et al., 2001; Trincao et al., 2001; Zhou et al., 2001) suggested that the ability to bypass damaged template bases arises from a very open and solvent-accessible active site, a feature that also allows highly error-prone DNA synthesis to occur. Subsequent Y-family polymerase structures have supported this original proposal and have also been able to identify a few protein-DNA interactions that facilitate bypass of specific DNA lesions, but given the wide range of specificities displayed by the Y-family polymerases, there are clearly more specificity determinants that remain to be discovered. "
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    ABSTRACT: Y-family polymerases help cells tolerate DNA damage by performing translesion synthesis opposite damaged DNA bases, yet they also have a high intrinsic error rate. We constructed chimeras of two closely related Y-family polymerases that display distinctly different activity profiles and found that the polypeptide linker that tethers the catalytic polymerase domain to the C-terminal DNA-binding domain is a major determinant of overall polymerase activity, nucleotide incorporation fidelity, and abasic site-bypass ability. Exchanging just 3 out of the 15 linker residues is sufficient to interconvert the polymerase activities tested. Crystal structures of four chimeras show that the conformation of the protein correlates with the identity of the interdomain linker sequence. Thus, residues that are more than 15 Å away from the active site are able to influence many aspects of polymerase activity by altering the relative orientations of the catalytic and DNA-binding domains.
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    • "These polymerases are functional even when DNA lesions are present since they can accommodate the lesions at their active sites, replicating across the lesion (11). Polη is a member of the Y-family polymerases which is able to replicate across UV lesions (12,13). Polη is recruited to the sites of replication and colocalizes with PCNA and Rad18 in foci upon UV irradiation (8,9,14). "
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