PIP domain ͉ UBZ domain ͉ PCNA ubiquitination ͉ exchange

Rev1, a Y family DNA polymerase (Pol) functions together with Pol, a B family Pol comprised of the Rev3 catalytic subunit and Rev7 accessory subunit, in promoting translesion DNA synthesis (TLS). Extensive genetic studies with Saccharomyces cerevisiae have indicated a requirement of both Pol and Rev1 for damage-induced mutagenesis, implicating their involvement in mutagenic TLS. Pol is specifically adapted to promote the extension step of lesion bypass, as it proficiently extends primer termini opposite DNA lesions, and it is also a proficient extender of mismatched primer termini on undamaged DNAs. Since TLS through UV-induced lesions and various other DNA lesions does not depend upon the DNA-synthetic activity of Rev1, Rev1 must contribute to Pol-dependent TLS in a nonenzymatic way. Here, we provide evidence for the physical association of Rev1 with Pol and show that this binding is mediated through the C terminus of Rev1 and the polymerase domain of Rev3. Importantly, a rev1 mutant that lacks the C-terminal 72 residues which inactivate interaction with Rev3 exhibits the same high degree of UV sensitivity and defectiveness in UV-induced mutagenesis as that conferred by the rev1⌬ mutation. We propose that Rev1 binding to Pol is indispensable for the targeting of Pol to the replication fork stalled at a DNA lesion. In addition to this structural role, Rev1 binding enhances the proficiency of Pol for the extension of mismatched primer termini on undamaged DNAs and for the extension of primer termini opposite DNA lesions.DNA lesions in the template strand block the progression of the replication fork. In Saccharomyces cerevisiae, the Rad6-Rad18 ubiquitin-conjugating enzyme complex (2, 3) promotes replication through DNA lesions via at least three different pathways that include translesion synthesis (TLS) by DNA polymerases (Pols) and and a Rad5-Mms2-Ubc13-dependent postreplication repair pathway (36).Pol is unique among eukaryotic TLS Pols in its proficiency for replication through UV-induced cyclobutane pyrimidine dimers (CPDs) in a relatively error-free manner (17,20,38,40). Inactivation of Pol in both yeast and humans confers enhanced UV-induced mutagenesis (35,37,41,42) and, in humans, causes a cancer-prone syndrome, the variant form of xeroderma pigmentosum (XPV) (15, 28). Although proficient replication through certain DNA lesions, such as CPDs, can be accomplished by one Pol, replication through many DNA lesions requires the concerted action of two Pols in which one Pol carries out the nucleotide insertion reaction opposite the lesion site and the other Pol performs the subsequent extension reaction (33, 34). Pol, comprised of the Rev3 catalytic subunit and the Rev7 accessory subunit (32), is highly specialized for performing the extension step of TLS (33,34).Extensive genetic studies with yeast have indicated a requirement of Pol in mutagenesis induced by DNA-damaging agents (18,22,25,26). For example, mutations in REV3 or REV7 cause a large reduction in the incidence of mutagenesis induced by UV radiatio...

DNA polymerase δ (Polδ) plays an essential role in replication from yeast to humans. Polδ in Saccharomyces cerevisiae is comprised of three subunits, the catalytic subunit Pol3 and the accessory subunits Pol31 and Pol32. Yeast Polδ exhibits a very high processivity in synthesizing DNA with the proliferating cell nuclear antigen (PCNA) sliding clamp; however, it has remained unclear how Polδ binds PCNA to achieve its high processivity. Here we show that PCNA interacting protein (PIP) motifs in all three subunits contribute to PCNA-stimulated DNA synthesis by Polδ, and mutational inactivation of all three PIP motifs abrogates its ability to synthesize DNA with PCNA. Genetic analyses of mutations in these PIPs have revealed that in the absence of functional Pol32 PIP domain, PCNA binding by both the Pol3 and Pol31 subunits becomes essential for cell viability. Based on our biochemical and genetic studies we infer that yeast Polδ can simultaneously utilize all three PIP motifs during PCNA-dependent DNA synthesis, and suggest that Polδ binds the PCNA homotrimer via its three subunits. We consider the implications of these observations for Polδ's role in DNA replication.

The Rev1 protein of Saccharomyces cerevisiae functions in translesion synthesis (TLS) together with DNA polymerase (Pol) , which is comprised of the Rev3 catalytic and the Rev7 accessory subunits. Rev1, a member of the Y family of Pols, differs from other members in its high degree of specificity for incorporating a C opposite template G as well as opposite an abasic site. Although Rev1 is indispensable for Pol-dependent TLS, its DNA synthetic activity is not required for many of the Pol-dependent lesion bypass events. This observation has suggested a structural role for Rev1 in this process. Here we show that in yeast, Rev1 forms a stable complex with Rev7, and the two proteins copurify. Importantly, the polymerase-associated domain (PAD) of Rev1 mediates its binding to Rev7. These observations reveal a novel role for the PAD region of Rev1 in protein-protein interactions, and they raise the possibility of a similar involvement of the PAD of other Y family Pols in protein-protein interactions. We discuss the possible roles of Rev1 versus the Rev1-Rev7 complex in TLS.Rad6, a ubiquitin-conjugating enzyme, exists in vivo in a tight complex with Rad18, which is a DNA binding protein (1, 2) and also acts as an E3 in the ubiquitin conjugation process (11). Rad6-Rad18-mediated ubiquitin conjugation promotes replication through DNA lesions via at least three different pathways: DNA polymerase (Pol) -dependent translesion synthesis (TLS), Pol dependent TLS, and a Rad5-Mms2-Ubc13-dependent pathway, the mechanism of which is not known (41).Pol promotes efficient and relatively error-free synthesis through UV-induced cyclobutane pyrimidine dimers which form at TT, TC, and CC sites (12,16,45,46); consequently, inactivation of Pol in Saccharomyces cerevisiae and human cells confers enhanced UV mutagenesis (13,30,39,44,47,48) and in humans causes the variant form of xeroderma pigmentosum (12,29). Pol, which is comprised of the Rev3 catalytic subunit and the Rev7 accessory subunit (33), is indispensable for UV mutagenesis in yeast (19,21,23) as well as human cells (6,18,24), and genetic studies in yeast have indicated its requirement for mutagenesis resulting from TLS occurring through abasic sites (14) and also through bases damaged upon treatment with certain chemical agents (35). Pol promotes lesion bypass primarily via its role as an extender, wherein following the insertion of a nucleotide opposite the DNA lesion by an inserter polymerase, Pol performs the extension of the nascent primer terminus (9, 15, 17, 37). For its role in TLS, however, Pol requires the Rev1 protein, which like the Rev3 and Rev7 proteins is indispensable for UV mutagenesis (19,20,22,23,36) and for mutagenesis resulting from TLS occurring through abasic sites (14) and through other damaged bases (3).Rev1, although a member of the Y family of DNA polymerases, differs from the other members in its specificity for predominantly inserting a C opposite template G and also opposite the other template nucleotides as well as an abasic site. Thus, Rev1 is a h...

In yeast, Rad6-Rad18-dependent lesion bypass involves translesion synthesis (TLS) by DNA polymerases h or z or Rad5-dependent postreplication repair (PRR) in which error-free replication through the DNA lesion occurs by template switching. Rad5 functions in PRR via its two distinct activities-a ubiquitin ligase that promotes Mms2-Ubc13-mediated K63-linked polyubiquitination of PCNA at its lysine 164 residue and a DNA helicase that is specialized for replication fork regression. Both these activities are important for Rad5's ability to function in PRR. Here we provide evidence for the requirement of Rad5 in TLS mediated by Polz. Using duplex plasmids carrying different site-specific DNA lesions-an abasic site, a cis-syn TT dimer, a (6-4) TT photoproduct, or a G-AAF adduct-we show that Rad5 is needed for Polz-dependent TLS. Rad5 action in this role is likely to be structural, since neither the inactivation of its ubiquitin ligase activity nor the inactivation of its helicase activity impairs its role in TLS.

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 C 2 H 2 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 C 2 H 2 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 C 2 H 2 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.Genetic and biochemical studies of Saccharomyces cerevisiae have indicated the requirement of the Rad6-Rad18 ubiquitin (Ub)-conjugating enzyme complex (1, 2) for promoting replication through DNA lesions which block synthesis by the replicative DNA polymerases (Pols). In yeast, Rad6-Rad18-dependent lesion bypass could occur via translesion DNA synthesis (TLS) by the action of Pol or Pol (20,22,25), or it may involve the Rad5-Mms2-Ubc13-dependent postreplicational repair of discontinuities that form in the newly synthesized DNA strand opposite from DNA lesions (27).Both in yeast and humans, PCNA plays a key role in modulating the various Rad6-Rad18-dependent lesion bypass processes. The TLS Pols, such as Pol from yeast and Pols , , and from humans, interact physically and functionally with PCNA, and PCNA binding is stimulatory to the catalytic activity of these Pols on undamaged and damaged DNAs (9)(10)(11)14). In addition, genetic studies of yeast have shown that PCNA binding is indispensable for the ability of Pol to function in TLS in vivo, since mutations in the PCNA binding PIP motif of Pol render cells as UV sensitive as those lacking Pol (11).Treatment of yeast or human cells with DNA-damaging agents elicits the monoubiquitination of PCNA at its lysine 164 residue, and subsequently, this residue becomes polyubiquitinated via a lysine 63-linked chain (16). Genetic studies have shown that PCNA monoubiquitination is mediated by the Rad6-Rad18 enzyme and that polyubiquitination requires the additional action of the Mms2-Ubc13-Rad5 enzyme complex (16), in which Rad5 provides the ubiquitin ligase function (28) and the Mms2-Ubc13 complex specifically promotes lysine 63-linked polyubiquitin chain formation (18). Also, genetic observations of yeast have indicated that PCNA monoubiquitination modulates t...

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Extracellular vesicles (EV), also known as membrane vesicles, are produced as an end product of secretion by both pathogenic and non-pathogenic bacteria. Several reports suggest that archaea, gram-negative bacteria, and eukaryotic cells secrete membrane vesicles as a means for cell-free intercellular communication. EVs influence intercellular communication by transferring a myriad of biomolecules including genetic information. Also, EVs have been implicated in many phenomena such as stress response, intercellular competition, lateral gene transfer, and pathogenicity. However, the cellular process of secreting EVs in gram-positive bacteria is less studied. A notion with the thick cell-walled microbes such as gram-positive bacteria is that the EV release is impossible among them. The role of gram-positive EVs in health and diseases is being studied gradually. Being nano-sized, the EVs from gram-positive bacteria carry a diversity of cargo compounds that have a role in bacterial competition, survival, invasion, host immune evasion, and infection. In this review, we summarise the current understanding of the EVs produced by gram-positive bacteria. Also, we discuss the functional aspects of these components while comparing them with gram-negative bacteria.