Structural basis of the 3′-end recognition of a leading strand in stalled replication forks by PriA

ABSTRACTHMCES can covalently crosslink to abasic sites in single-stranded DNA at stalled replication forks to prevent genome instability. Here, we report crystal structures of the HMCES SRAP domain in complex with DNA-damage substrates, revealing interactions with both single-stranded and duplex segments of 3’ overhang DNA. HMCES may also bind gapped DNA and 5’ overhang structures to align single stranded abasic sites for crosslinking to the conserved Cys2 of its catalytic triad.

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Cryo-EM structure of human Pol κ bound to DNA and mono-ubiquitylated PCNA

Nature Communications ◽

10.1038/s41467-021-26251-6 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Claudia Lancey ◽

Muhammad Tehseen ◽

Souvika Bakshi ◽

Matthew Percival ◽

Masateru Takahashi ◽

...

Keyword(s):

Dna Damage ◽

Zinc Fingers ◽

Structural Basis ◽

Replication Forks ◽

Catalytic Core ◽

Dna Templates ◽

Secondary Interaction ◽

Ubiquitin Binding ◽

Y Family ◽

Stalled Replication Forks

AbstractY-family DNA polymerase κ (Pol κ) can replicate damaged DNA templates to rescue stalled replication forks. Access of Pol κ to DNA damage sites is facilitated by its interaction with the processivity clamp PCNA and is regulated by PCNA mono-ubiquitylation. Here, we present cryo-EM reconstructions of human Pol κ bound to DNA, an incoming nucleotide, and wild type or mono-ubiquitylated PCNA (Ub-PCNA). In both reconstructions, the internal PIP-box adjacent to the Pol κ Polymerase-Associated Domain (PAD) docks the catalytic core to one PCNA protomer in an angled orientation, bending the DNA exiting the Pol κ active site through PCNA, while Pol κ C-terminal domain containing two Ubiquitin Binding Zinc Fingers (UBZs) is invisible, in agreement with disorder predictions. The ubiquitin moieties are partly flexible and extend radially away from PCNA, with the ubiquitin at the Pol κ-bound protomer appearing more rigid. Activity assays suggest that, when the internal PIP-box interaction is lost, Pol κ is retained on DNA by a secondary interaction between the UBZs and the ubiquitins flexibly conjugated to PCNA. Our data provide a structural basis for the recruitment of a Y-family TLS polymerase to sites of DNA damage.

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Schizosaccharomyces pombe Swi1, Swi3, and Hsk1 Are Components of a Novel S-Phase Response Pathway to Alkylation Damage

Molecular and Cellular Biology ◽

10.1128/mcb.25.7.2770-2784.2005 ◽

2005 ◽

Vol 25 (7) ◽

pp. 2770-2784 ◽

Cited By ~ 60

Author(s):

Elena Sommariva ◽

Till K. Pellny ◽

Nilay Karahan ◽

Sanjay Kumar ◽

Joel A. Huberman ◽

...

Keyword(s):

Schizosaccharomyces Pombe ◽

S Phase ◽

Replication Forks ◽

Replication Process ◽

Alkylation Damage ◽

Checkpoint Response ◽

Checkpoint Pathway ◽

Mating Type Switching ◽

Leading Strand ◽

Stalled Replication Forks

ABSTRACT The Swi1 and Swi3 proteins are required for mat1 imprinting and mating-type switching in Schizosaccharomyces pombe, where they mediate a pause of leading-strand replication in response to a lagging-strand signal. In addition, Swi1 has been demonstrated to be involved in the checkpoint response to stalled replication forks, as was described for the Saccharomyces cerevisiae homologue Tof1. This study addresses the roles of Swi1 and Swi3 during a replication process perturbed by the presence of template bases alkylated by methyl methanesulfonate (MMS). Both the swi1 and swi3 mutations have additive effects on MMS sensitivity and on the MMS-induced damage checkpoint response when combined with chk1 and cds1, but they are nonadditive with hsk1. Cells with swi1, swi3, or hsk1 mutations are also defective in slowing progression through S phase in response to MMS damage. Moreover, swi1 and swi3 strains show increased levels of genomic instability even in the absence of exogenously induced DNA damage. Chromosome fragmentation, increased levels of single-stranded DNA, increased recombination, and instability of replication forks stalled in the presence of hydroxyurea are observed, consistent with the possibility that the replication process is affected in these mutants. In conclusion, Swi1, Swi3, and Hsk1 act in a novel S-phase checkpoint pathway that contributes to replication fork maintenance and to survival of alkylation damage.

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Both DNA Polymerases δ and ε Contact Active and Stalled Replication Forks Differently

Molecular and Cellular Biology ◽

10.1128/mcb.00190-17 ◽

2017 ◽

Vol 37 (21) ◽

Cited By ~ 3

Author(s):

Chuanhe Yu ◽

Haiyun Gan ◽

Zhiguo Zhang

Keyword(s):

Dna Synthesis ◽

Dna Polymerase ◽

Genome Duplication ◽

Dna Polymerases ◽

Distinct Pattern ◽

Replication Forks ◽

Okazaki Fragments ◽

Dna Replication Stress ◽

Leading Strand ◽

Stalled Replication Forks

ABSTRACT Three DNA polymerases, polymerases α, δ, and ε (Pol α, Pol δ, and Pol ε), are responsible for eukaryotic genome duplication. When DNA replication stress is encountered, DNA synthesis stalls until the stress is ameliorated. However, it is not known whether there is a difference in the association of each polymerase with active and stalled replication forks. Here, we show that each DNA polymerase has a distinct pattern of association with active and stalled replication forks. Pol α is enriched at extending Okazaki fragments of active and stalled forks. In contrast, although Pol δ contacts the nascent lagging strands of active and stalled forks, it binds to only the matured (and not elongating) Okazaki fragments of stalled forks. Pol ε has greater contact with the nascent single-stranded DNA (ssDNA) of the leading strand on active forks than on stalled forks. We propose that the configuration of DNA polymerases at stalled forks facilitates the resumption of DNA synthesis after stress removal.

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Both DNA Polymerases δ and ε Contact Active and Stalled Replication Forks differently

10.1101/170795 ◽

2017 ◽

Author(s):

Chuanhe Yu ◽

Haiyun Gan ◽

Zhiguo Zhang

Keyword(s):

Dna Replication ◽

Dna Synthesis ◽

Dna Polymerase ◽

Genome Duplication ◽

Dna Polymerases ◽

Replication Forks ◽

Okazaki Fragments ◽

Dna Replication Stress ◽

Leading Strand ◽

Stalled Replication Forks

AbstractThree DNA polymerases (Pol α, Pol δ, and Pol ε) are responsible for eukaryotic genome duplication. When DNA replication stress is encountered, DNA synthesis stalls until the stress is ameliorated. However, it is not known whether there is a difference in the association of each polymerase with active and stalled replication forks. Here, we show that each DNA polymerase has distinct patterns of association with active and stalled replication forks. Pol α is enriched at extending Okazaki fragments of active and stalled forks. In contrast, although Pol δ contacts the nascent lagging strands of active and stalled forks, it binds to only the matured (and not elongating) Okazaki fragments of stalled forks. Pol ε has a greater contact with the nascent ssDNA of leading strand on active forks compared with stalled forks. We propose that the configuration of DNA polymerases at stalled forks facilitate resumption of DNA synthesis after stress removal.

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