Tunability of DNA Polymerase Stability during Eukaryotic DNA Replication

Lewis, Jacob S.; Spenkelink, Lisanne M.; Schauer, Grant D.; Yurieva, Olga; Mueller, S.H.; Natarajan, Varsha; Kaur, Gurleen; Maher, Claire; Kay, Callum; O'Donnell, Mike; Oijen, Antoine M. van

doi:10.1016/j.molcel.2019.10.005

Cited by 88 publications

(119 citation statements)

References 51 publications

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“…7, P = 0.0007). Taken together, the modest effect of Rad18 knockdown vs. the strong effect of Pol k knockdown suggests that Pol k may also have a fork restart function independent of Rad18 (121)(122)(123). Surprisingly, HU treatment did not augment the effect of Pol k knockdown (Fig.…”

Section: Translesion Dna Synthesis Enzymes Stabilize (Ctg/cag)100 Agamentioning

confidence: 85%

Replication stress at microsatellites causes DNA double-strand breaks and break-induced replication

Gadgil

Romer

Goodman

et al. 2020

Journal of Biological Chemistry

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Short tandemly repeated DNA sequences, termed microsatellites, are abundant in the human genome. These microsatellites exhibit length instability and susceptibility to DNA double strand breaks (DSBs) due to their tendency to form stable non-B DNA structures. Replication-dependent microsatellite DSBs are linked to genome instability signatures in human developmental diseases and cancers. To probe the causes and consequences of microsatellite DSBs, we designed a dual fluorescence reporter system to detect DSBs at expanded (CTG/CAG)n and polypurine/polypyrimidine (Pu/Py) mirror repeat structures alongside the c-myc replication origin integrated at a single ectopic chromosomal site. Restriction cleavage near the (CTG/CAG)100 microsatellite leads to homology directed single strand annealing between flanking AluY elements, and reporter gene deletion that can be detected by flow cytometry. However, in the absence of restriction cleavage, endogenous and exogenous replication stressors induce DSBs at the (CTG/CAG)100 and Pu/Py microsatellites. DSBs map to a narrow region at the downstream edge of the (CTG)100 lagging strand template. (CTG/CAG)n chromosome fragility is repeat length dependent, while instability at the (Pu/Py) microsatellites depends on replication polarity. Strikingly, restriction-generated DSBs and replication-dependent DSBs are not repaired by the same mechanism. Knockdown of DNA damage response proteins increase (Rad18, Pol η, Pol κ) or decrease (Mus81) the sensitivity of the (CTG/CAG)100 microsatellites to replication stress. Replication stress and DSBs at the ectopic (CTG/CAG)100 microsatellite lead to break induced replication and high frequency mutagenesis at a flanking thymidine kinase gene. Our results show that non-B structure-prone microsatellites are susceptible to replication-dependent DSBs that cause genome instability.

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Section: Translesion Dna Synthesis Enzymes Stabilize (Ctg/cag)100 Agamentioning

confidence: 85%

Replication stress at microsatellites causes DNA double-strand breaks and break-induced replication

Gadgil

Romer

Goodman

et al. 2020

Journal of Biological Chemistry

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“…Therefore, an intriguing possibility for the unused C-terminal consensus Pol32 PIP motif is to bind Pol α-primase. Supporting this suggestion, S.c. Pol32 has been demonstrated to interact with Pol α-primase (46,47), and Pol31 was recently shown to help retain Pol δ in the replisome during multiple cycles of Okazaki fragment synthesis, supporting a connection between Pol32 and Pol α-primase during replisome function (48). Recent structural studies reveal that one Pol α-primase is anchored to the replisome by the Ctf4 trimer (49), and thus the Pol α-primase-Pol32 interaction likely recruits Pol δ to the replication fork.…”

Section: Possible Function Of the 4fe-4s Cluster In Sensing Intracellmentioning

confidence: 98%

Structure of eukaryotic DNA polymerase δ bound to the PCNA clamp while encircling DNA

Zheng

Georgescu

et al. 2020

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

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The DNA polymerase (Pol) δ ofSaccharomyces cerevisiae(S.c.) is composed of the catalytic subunit Pol3 along with two regulatory subunits, Pol31 and Pol32. Pol δ binds to proliferating cell nuclear antigen (PCNA) and functions in genome replication, repair, and recombination. Unique among DNA polymerases, the Pol3 catalytic subunit contains a 4Fe-4S cluster that may sense the cellular redox state. Here we report the 3.2-Å cryo-EM structure of S.c. Pol δ in complex with primed DNA, an incoming ddTTP, and the PCNA clamp. Unexpectedly, Pol δ binds only one subunit of the PCNA trimer. This singular yet extensive interaction holds DNA such that the 2-nm-wide DNA threads through the center of the 3-nm interior channel of the clamp without directly contacting the protein. Thus, a water-mediated clamp and DNA interface enables the PCNA clamp to “waterskate” along the duplex with minimum drag. Pol31 and Pol32 are positioned off to the side of the catalytic Pol3-PCNA-DNA axis. We show here that Pol31-Pol32 binds single-stranded DNA that we propose underlies polymerase recycling during lagging strand synthesis, in analogy toEscherichia colireplicase. Interestingly, the 4Fe-4S cluster in the C-terminal CysB domain of Pol3 forms the central interface to Pol31-Pol32, and this strategic location may explain the regulation of the oxidation state on Pol δ activity, possibly useful during cellular oxidative stress. Importantly, human cancer and other disease mutations map to nearly every domain of Pol3, suggesting that all aspects of Pol δ replication are important to human health and disease.

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“…ukaryotic replisomes are multiprotein complexes consisting, minimally, of the CMG helicase [MCM2-7 (M), CDC45 (C), and GINS (go, ichi, ni, san) proteins (G)], which forms a ring around the leading strand template. Other components include the pol α, ε, and δ polymerases, MCM10, and a few accessory factors [1][2][3][4][5][6][7] . The identification and characterization of the minimal components of biochemically active replisomes, the result of decades of extraordinary work from multiple laboratories, necessarily reflects studies with deproteinized model DNA substrates under carefully controlled conditions.…”

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confidence: 99%

DONSON and FANCM associate with different replisomes distinguished by replication timing and chromatin domain

et al. 2020

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Duplication of mammalian genomes requires replisomes to overcome numerous impediments during passage through open (eu) and condensed (hetero) chromatin. Typically, studies of replication stress characterize mixed populations of challenged and unchallenged replication forks, averaged across S phase, and model a single species of "stressed" replisome. Here, in cells containing potent obstacles to replication, we find two different lesion proximal replisomes. One is bound by the DONSON protein and is more frequent in early S phase, in regions marked by euchromatin. The other interacts with the FANCM DNA translocase, is more prominent in late S phase, and favors heterochromatin. The two forms can also be detected in unstressed cells. ChIP-seq of DNA associated with DONSON or FANCM confirms the bias of the former towards regions that replicate early and the skew of the latter towards regions that replicate late.

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Tunability of DNA Polymerase Stability during Eukaryotic DNA Replication

Cited by 88 publications

References 51 publications

Replication stress at microsatellites causes DNA double-strand breaks and break-induced replication

Replication stress at microsatellites causes DNA double-strand breaks and break-induced replication

Structure of eukaryotic DNA polymerase δ bound to the PCNA clamp while encircling DNA

DONSON and FANCM associate with different replisomes distinguished by replication timing and chromatin domain

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