scholarly journals Complex Formation of Yeast Rev1 and Rev7 Proteins: a Novel Role for the Polymerase-Associated Domain

2005 ◽  
Vol 25 (21) ◽  
pp. 9734-9740 ◽  
Author(s):  
Narottam Acharya ◽  
Lajos Haracska ◽  
Robert E. Johnson ◽  
Ildiko Unk ◽  
Satya Prakash ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Protein Interactions ◽  
Synthetic Activity ◽  
Stable Complex ◽  
Abasic Site ◽  
Protein Protein Interactions ◽  
Lesion Bypass ◽  
Accessory Subunits ◽  
High Degree ◽  
Y Family

ABSTRACT The Rev1 protein of Saccharomyces cerevisiae functions in translesion synthesis (TLS) together with DNA polymerase (Pol) ζ, 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 Polζ-dependent TLS, its DNA synthetic activity is not required for many of the Polζ-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.

scholarly journals Complex Formation with Rev1 Enhances the Proficiency of Saccharomyces cerevisiae DNA Polymerase ζ for Mismatch Extension and for Extension Opposite from DNA Lesions

2006 ◽  
Vol 26 (24) ◽  
pp. 9555-9563 ◽  
Author(s):  
Narottam Acharya ◽  
Robert E. Johnson ◽  
Satya Prakash ◽  
Louise Prakash
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Polymerase ◽  
Dna Lesions ◽  
Synthetic Activity ◽  
Lesion Bypass ◽  
Dna Lesion ◽  
C Terminus ◽  
Accessory Subunit ◽  
Induced Mutagenesis ◽  
Y Family

ABSTRACT Rev1, a Y family DNA polymerase (Pol) functions together with Polζ, 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 Polζ and Rev1 for damage-induced mutagenesis, implicating their involvement in mutagenic TLS. Polζ 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 Polζ-dependent TLS in a nonenzymatic way. Here, we provide evidence for the physical association of Rev1 with Polζ 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 rev1Δ mutation. We propose that Rev1 binding to Polζ is indispensable for the targeting of Polζ to the replication fork stalled at a DNA lesion. In addition to this structural role, Rev1 binding enhances the proficiency of Polζ for the extension of mismatched primer termini on undamaged DNAs and for the extension of primer termini opposite DNA lesions.

scholarly journals E. coli primase and DNA polymerase III holoenzyme are able to bind concurrently to a primed template during DNA replication

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Andrea Bogutzki ◽  
Natalie Naue ◽  
Lidia Litz ◽  
Andreas Pich ◽  
Ute Curth
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Protein Interactions ◽  
Dna Polymerase Iii ◽  
Protein Protein Interactions ◽  
Single Stranded Dna ◽  
E Coli ◽  
Polymerase Iii ◽  
C Terminus ◽  
Pol Iii

Abstract During DNA replication in E. coli, a switch between DnaG primase and DNA polymerase III holoenzyme (pol III) activities has to occur every time when the synthesis of a new Okazaki fragment starts. As both primase and the χ subunit of pol III interact with the highly conserved C-terminus of single-stranded DNA-binding protein (SSB), it had been proposed that the binding of both proteins to SSB is mutually exclusive. Using a replication system containing the origin of replication of the single-stranded DNA phage G4 (G4ori) saturated with SSB, we tested whether DnaG and pol III can bind concurrently to the primed template. We found that the addition of pol III does not lead to a displacement of primase, but to the formation of higher complexes. Even pol III-mediated primer elongation by one or several DNA nucleotides does not result in the dissociation of DnaG. About 10 nucleotides have to be added in order to displace one of the two primase molecules bound to SSB-saturated G4ori. The concurrent binding of primase and pol III is highly plausible, since even the SSB tetramer situated directly next to the 3′-terminus of the primer provides four C-termini for protein-protein interactions.

scholarly journals Detection of Protein-Protein Interactions in the Alkanesulfonate Monooxygenase System from Escherichia coli

2006 ◽  
Vol 188 (23) ◽  
pp. 8153-8159 ◽  
Author(s):  
Kholis Abdurachim ◽  
Holly R. Ellis
Keyword(s):  
Circular Dichroism ◽  
Protein Interactions ◽  
Visible Region ◽  
Circular Dichroism Spectroscopy ◽  
Flavin Mononucleotide ◽  
Stable Complex ◽  
Monooxygenase System ◽  
Protein Protein Interactions ◽  
Alkanesulfonate Monooxygenase ◽  
Circular Dichroïsm

ABSTRACT The two-component alkanesulfonate monooxygenase system utilizes reduced flavin as a substrate to catalyze a unique desulfonation reaction during times of sulfur starvation. The importance of protein-protein interactions in the mechanism of flavin transfer was analyzed in these studies. The results from affinity chromatography and cross-linking experiments support the formation of a stable complex between the flavin mononucleotide (FMN) reductase (SsuE) and monooxygenase (SsuD). Interactions between the two proteins do not lead to overall conformational changes in protein structure, as indicated by the results from circular dichroism spectroscopy in the far-UV region. However, subtle changes in the flavin environment of FMN-bound SsuE that occur in the presence of SsuD were identified by circular dichroism spectroscopy in the visible region. These data are supported by the results from fluorescent spectroscopy experiments, where a dissociation constant of 0.0022 ± 0.0010 μM was obtained for the binding of SsuE to SsuD. Based on these studies, the stoichiometry for protein-protein interactions is proposed to involve a 1:1 monomeric association of SsuE with SsuD.

scholarly journals Mechanism of double-base lesion bypass catalyzed by a Y-family DNA polymerase

2008 ◽  
Vol 36 (12) ◽  
pp. 3867-3878 ◽  
Author(s):  
Jessica A. Brown ◽  
Sean A. Newmister ◽  
Kevin A. Fiala ◽  
Zucai Suo
Keyword(s):  
Dna Polymerase ◽  
Lesion Bypass ◽  
Double Base ◽  
Base Lesion ◽  
Y Family

scholarly journals Multisite SUMOylation restrains DNA polymerase η interactions with DNA damage sites

2020 ◽  
Vol 295 (25) ◽  
pp. 8350-8362 ◽  
Author(s):  
Claire Guérillon ◽  
Stine Smedegaard ◽  
Ivo A. Hendriks ◽  
Michael L. Nielsen ◽  
Niels Mailand
Keyword(s):  
Dna Damage ◽  
Dna Polymerase ◽  
Feedback Inhibition ◽  
Dna Lesions ◽  
Nuclear Antigen ◽  
Inhibition Mechanism ◽  
Proteomic Profiling ◽  
Lesion Bypass ◽  
Y Family ◽  
Damaged Dna

Translesion DNA synthesis (TLS) mediated by low-fidelity DNA polymerases is an essential cellular mechanism for bypassing DNA lesions that obstruct DNA replication progression. However, the access of TLS polymerases to the replication machinery must be kept tightly in check to avoid excessive mutagenesis. Recruitment of DNA polymerase η (Pol η) and other Y-family TLS polymerases to damaged DNA relies on proliferating cell nuclear antigen (PCNA) monoubiquitylation and is regulated at several levels. Using a microscopy-based RNAi screen, here we identified an important role of the SUMO modification pathway in limiting Pol η interactions with DNA damage sites in human cells. We found that Pol η undergoes DNA damage- and protein inhibitor of activated STAT 1 (PIAS1)-dependent polySUMOylation upon its association with monoubiquitylated PCNA, rendering it susceptible to extraction from DNA damage sites by SUMO-targeted ubiquitin ligase (STUbL) activity. Using proteomic profiling, we demonstrate that Pol η is targeted for multisite SUMOylation, and that collectively these SUMO modifications are essential for PIAS1- and STUbL-mediated displacement of Pol η from DNA damage sites. These findings suggest that a SUMO-driven feedback inhibition mechanism is an intrinsic feature of TLS-mediated lesion bypass functioning to curtail the interaction of Pol η with PCNA at damaged DNA to prevent harmful mutagenesis.

Protein interactions in T7 DNA replisome inhibit the bypass of abasic site by DNA polymerase

Mutagenesis ◽  
2019 ◽  
Author(s):  
Zhenyu Zou ◽  
Tingting Liang ◽  
Zhongyan Xu ◽  
Jiayu Xie ◽  
Shuming Zhang ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Protein Interactions ◽  
Accessory Proteins ◽  
Abasic Site ◽  
Traditional Concept ◽  
Translesion Dna Synthesis ◽  
Dna Lesion ◽  
Translesion Dna

Abstract Abasic site as a common DNA lesion blocks DNA replication and is highly mutagenic. Protein interactions in T7 DNA replisome facilitate DNA replication and translesion DNA synthesis. However, bypass of an abasic site by T7 DNA replisome has never been investigated. In this work, we used T7 DNA replisome and T7 DNA polymerase alone as two models to study DNA replication on encountering an abasic site. Relative to unmodified DNA, abasic site strongly inhibited primer extension and completely blocked strand-displacement DNA synthesis, due to the decreased fraction of enzyme–DNA productive complex and the reduced average extension rates. Moreover, abasic site at DNA fork inhibited the binding of DNA polymerase or helicase onto fork and the binding between polymerase and helicase at fork. Notably and unexpectedly, we found DNA polymerase alone bypassed an abasic site on primer/template (P/T) substrate more efficiently than did polymerase and helicase complex bypass it at fork. The presence of gp2.5 further inhibited the abasic site bypass at DNA fork. Kinetic analysis showed that this inhibition at fork relative to that on P/T was due to the decreased fraction of productive complex instead of the average extension rates. Therefore, we found that protein interactions in T7 DNA replisome inhibited the bypass of DNA lesion, different from all the traditional concept that protein interactions or accessory proteins always promote DNA replication and DNA damage bypass, providing new insights in translesion DNA synthesis performed by DNA replisome.

scholarly journals Efficient and Error-Free Replication past a Minor-Groove N2-Guanine Adduct by the Sequential Action of Yeast Rev1 and DNA Polymerase ζ

2004 ◽  
Vol 24 (16) ◽  
pp. 6900-6906 ◽  
Author(s):  
M. Todd Washington ◽  
Irina G. Minko ◽  
Robert E. Johnson ◽  
Lajos Haracska ◽  
Thomas M. Harris ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Close Contact ◽  
Dna Polymerases ◽  
Minor Groove ◽  
Synthetic Activity ◽  
Oxidation Reactions ◽  
Lesion Bypass ◽  
Dna Minor Groove

ABSTRACT Rev1, a member of the Y family of DNA polymerases, functions in lesion bypass together with DNA polymerase ζ (Polζ). Rev1 is a highly specialized enzyme in that it incorporates only a C opposite template G. While Rev1 plays an indispensable structural role in Polζ-dependent lesion bypass, the role of its DNA synthetic activity in lesion bypass has remained unclear. Since interactions of DNA polymerases with the DNA minor groove contribute to the nearly equivalent efficiencies and fidelities of nucleotide incorporation opposite each of the four template bases, here we examine the possibility that unlike other DNA polymerases, Rev1 does not come into close contact with the minor groove of the incipient base pair, and that enables it to incorporate a C opposite the N 2-adducted guanines in DNA. To test this idea, we examined whether Rev1 could incorporate a C opposite the γ-hydroxy-1,N 2-propano-2′deoxyguanosine DNA minor-groove adduct, which is formed from the reaction of acrolein with the N 2 of guanine. Acrolein, an α,β-unsaturated aldehyde, is generated in vivo as the end product of lipid peroxidation and from other oxidation reactions. We show here that Rev1 efficiently incorporates a C opposite this adduct from which Polζ subsequently extends, thereby completing the lesion bypass reaction. Based upon these observations, we suggest that an important role of the Rev1 DNA synthetic activity in lesion bypass is to incorporate a C opposite the various N 2-guanine DNA minor-groove adducts that form in DNA.

scholarly journals Mechanism of Abasic Lesion Bypass Catalyzed by a Y-family DNA Polymerase

2007 ◽  
Vol 282 (11) ◽  
pp. 8188-8198 ◽  
Author(s):  
Kevin A. Fiala ◽  
Cameron D. Hypes ◽  
Zucai Suo
Keyword(s):  
Dna Polymerase ◽  
Lesion Bypass ◽  
Y Family
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