The C-terminus of Dpb2 is required for interaction with Pol2 and for cell viability

Isoz, Isabelle; Persson, Ulla; Volkov, Kirill V.; Johansson, Erik

doi:10.1093/nar/gks880

Cited by 25 publications

(17 citation statements)

References 53 publications

(74 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Interestingly, the impaired Pol Dpb2-100p complex had normal in vitro polymerase activity, processivity and exonuclease activity. These data are consistent with earlier results from yeast and H. sapiens cells, where purified Pol2/Dpb3/Dpb4 complexes devoid of Dpb2p had biochemical properties indistinguishable from those of the wild-type Pol [49,86].…”

Section: Discussionsupporting

confidence: 93%

See 1 more Smart Citation

Fidelity consequences of the impaired interaction between DNA polymerase epsilon and the GINS complex

et al. 2015

View full text Add to dashboard Cite

DNA polymerase epsilon interacts with the CMG (Cdc45-MCM-GINS) complex by Dpb2p, the non-catalytic subunit of DNA polymerase epsilon. It is postulated that CMG is responsible for targeting of Pol ɛ to the leading strand. We isolated a mutator dpb2-100 allele which encodes the mutant form of Dpb2p. We showed previously that Dpb2-100p has impaired interactions with Pol2p, the catalytic subunit of Pol ɛ. Here, we present that Dpb2-100p has strongly impaired interaction with the Psf1 and Psf3 subunits of the GINS complex. Our in vitro results suggest that while dpb2-100 does not alter Pol ɛ's biochemical properties including catalytic efficiency, processivity or proofreading activity - it moderately decreases the fidelity of DNA synthesis. As the in vitro results did not explain the strong in vivo mutator effect of the dpb2-100 allele we analyzed the mutation spectrum in vivo. The analysis of the mutation rates in the dpb2-100 mutant indicated an increased participation of the error-prone DNA polymerase zeta in replication. However, even in the absence of Pol ζ activity the presence of the dpb2-100 allele was mutagenic, indicating that a significant part of mutagenesis is Pol ζ-independent. A strong synergistic mutator effect observed for transversions in the triple mutant dpb2-100 pol2-4 rev3Δ as compared to pol2-4 rev3Δ and dpb2-100 rev3Δ suggests that in the presence of the dpb2-100 allele the number of replication errors is enhanced. We hypothesize that in the dpb2-100 strain, where the interaction between Pol ɛ and GINS is weakened, the access of Pol δ to the leading strand may be increased. The increased participation of Pol δ on the leading strand in the dpb2-100 mutant may explain the synergistic mutator effect observed in the dpb2-100 pol3-5DV double mutant.

show abstract

Section: Discussionsupporting

confidence: 93%

“…A few years ago we found that Dpb2p, a non-catalytic subunit of DNA Pol , is important for faithful replication of genomic DNA [47,48]. It has been established that the C-terminus of Dpb2p is important for its interaction with Pol2p [49]. Isolated in our laboratory dpb2 mutants exhibit temperature-sensitivity and mutator phenotype [47,48].…”

Section: Introductionmentioning

confidence: 89%

Fidelity consequences of the impaired interaction between DNA polymerase epsilon and the GINS complex

et al. 2015

View full text Add to dashboard Cite

show abstract

“…One potential drawback to a stable connection between Pol e and CMG is the need for dynamic exchange between high-fidelity and lowfidelity polymerases to overcome template blocks during fork progression (41). Temperature-sensitive mutants of the Dpb2 subunit of Pol e that no longer bind stably to the Pol2 catalytic subunit confer a strong mutator phenotype that is partially dependent on Pol ζ, suggesting that low-fidelity polymerases have greater access to the leading strand when the CMG-Dpb2-Pol2 connection is disrupted (34,42,43). Together, these data suggest a model whereby the Pol e-CMG connection is flexible, enabling translesion and other polymerases to bind the DNA temporarily without displacing Pol e from the replisome.…”

Section: Discussionmentioning

confidence: 99%

“…The Dpb2 subunit of Pol e binds to both the catalytic subunit Pol2 and to the Psf1 subunit of GINS and, whereas this connection has been shown to be required to recruit Pol e to the replication fork, it was not clear whether it is essential for ongoing Pol e function with CMG during elongation (19,30,32,33). Indeed, a three-subunit form of Pol e lacking Dpb2 was shown to be a fully functional and processive polymerase on singly primed circular substrates so Dpb2 is not required for the polymerase activity of Pol e (34,35). To investigate the role of Dpb2 in Pol e function with CMG at a replication fork, we separately purified Dpb2 and the Pol2 catalytic subunit (Fig.…”

Section: The Dpb2 Subunit Of Pol E Is Required For Efficient Leading-mentioning

confidence: 99%

CMG helicase and DNA polymerase ε form a functional 15-subunit holoenzyme for eukaryotic leading-strand DNA replication

Langston

Zhang

Yurieva

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

144

161

View full text Add to dashboard Cite

DNA replication in eukaryotes is asymmetric, with separate DNA polymerases (Pol) dedicated to bulk synthesis of the leading and lagging strands. Pol α/primase initiates primers on both strands that are extended by Pol e on the leading strand and by Pol δ on the lagging strand. The CMG (Cdc45-MCM-GINS) helicase surrounds the leading strand and is proposed to recruit Pol e for leading-strand synthesis, but to date a direct interaction between CMG and Pol e has not been demonstrated. While purifying CMG helicase overexpressed in yeast, we detected a functional complex between CMG and native Pol e. Using pure CMG and Pol e, we reconstituted a stable 15-subunit CMG-Pol e complex and showed that it is a functional polymerase-helicase on a model replication fork in vitro. On its own, the Pol2 catalytic subunit of Pol e is inefficient in CMG-dependent replication, but addition of the Dpb2 protein subunit of Pol e, known to bind the Psf1 protein subunit of CMG, allows stable synthesis with CMG. Dpb2 does not affect Pol δ function with CMG, and thus we propose that the connection between Dpb2 and CMG helps to stabilize Pol e on the leading strand as part of a 15-subunit leading-strand holoenzyme we refer to as CMGE. Direct binding between Pol e and CMG provides an explanation for specific targeting of Pol e to the leading strand and provides clear mechanistic evidence for how strand asymmetry is maintained in eukaryotes.DNA replication | replication fork | helicase | polymerase | CMG

show abstract

“…The first module is the Pol2 N-terminal catalytic domain, which is dispensable for viability (26,27). The second module is the noncatalytic portion of the assembly comprising the essential Pol2 C-terminal domain (a catalytically defunct polymerase), Dpb2 (a defunct exonuclease), and the Dpb3 and Dpb4 ancillary factors (28)(29)(30)(31)(32)(33). Coordinated action of the CMG and Pol epsilon supports leading-strand synthesis (14,(34)(35)(36), and emerging evidence indicates that the noncatalytic module of Pol epsilon plays a separate role in replication, being essential for CMG formation in cells and perhaps by stimulating the DNA unwinding function of the CMG helicase (14,28,33,37,38).…”

mentioning

confidence: 99%

CMG–Pol epsilon dynamics suggests a mechanism for the establishment of leading-strand synthesis in the eukaryotic replisome

Zhou

Janska

Goswami

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

108

View full text Add to dashboard Cite

The replisome unwinds and synthesizes DNA for genome duplication. In eukaryotes, the Cdc45-MCM-GINS (CMG) helicase and the leading-strand polymerase, Pol epsilon, form a stable assembly. The mechanism for coupling DNA unwinding with synthesis is starting to be elucidated, however the architecture and dynamics of the replication fork remain only partially understood, preventing a molecular understanding of chromosome replication. To address this issue, we conducted a systematic single-particle EM study on multiple permutations of the reconstituted CMG-Pol epsilon assembly. Pol epsilon contains two flexibly tethered lobes. The noncatalytic lobe is anchored to the motor of the helicase, whereas the polymerization domain extends toward the side of the helicase. We observe two alternate configurations of the DNA synthesis domain in the CMG-bound Pol epsilon. We propose that this conformational switch might control DNA template engagement and release, modulating replisome progression.DNA replication | CMG helicase | DNA polymerase | single-particle electron microscopy D NA replication is catalyzed by the replisome, a molecular machine that coordinates DNA unwinding and synthesis (1). These two functions must be tightly coordinated to prevent the rise of genome instability, which is a major cause of cancer. DNA unwinding by a replicative helicase involves single-strand translocation of a hexameric motor, whereas DNA synthesis requires template priming by a primase and extension by dedicated replicative DNA polymerases (2). In eukaryotes, the helicase function is performed by the Cdc45-MCM-GINS (CMG) complex (3, 4) and the primase function is played by Pol alpha (5), whereas DNA synthesis is catalyzed by two specialized DNA polymerases, Pol epsilon and delta. According to the consensus view, Pol epsilon synthesizes the leading and Pol delta the lagging strand (6-11). However, recent studies indicate that the division of labor between replicative polymerases might be more promiscuous than originally thought (12, 13). In in vitroreconstituted DNA replication reactions, Pol delta can support leading-strand duplication (11, 14), but switching from Pol delta to epsilon is necessary for efficient establishment of leadingstrand synthesis (14). The mechanism of substrate handoff between the two polymerases is currently unknown.Recent breakthroughs in structural biology begin to provide an architectural framework to understand the interaction between helicase and polymerases at the replication fork. For example, studies on the CMG helicase and its subcomplexes have established that the MCM is a six-member ring with an N-terminal domain that serves as a processivity collar (15) and a C-terminal ATPase motor domain that provides the DNA unwinding function (16-21). High-resolution cryo-EM analysis has shown that the ATPase motor translocates on the leading-strand template (22), in agreement with work on Xenopus embryo extracts (23). The GINS and Cdc45 components of the CMG bind to the side and stabilize the N-terminal domain of the...

show abstract

The C-terminus of Dpb2 is required for interaction with Pol2 and for cell viability

Cited by 25 publications

References 53 publications

Fidelity consequences of the impaired interaction between DNA polymerase epsilon and the GINS complex

Fidelity consequences of the impaired interaction between DNA polymerase epsilon and the GINS complex

CMG helicase and DNA polymerase ε form a functional 15-subunit holoenzyme for eukaryotic leading-strand DNA replication

CMG–Pol epsilon dynamics suggests a mechanism for the establishment of leading-strand synthesis in the eukaryotic replisome

Contact Info

Product

Resources

About