Compartmentalization of the replication fork by single-stranded DNA binding protein regulates translesion synthesis

AbstractDNA replication is mediated by the coordinated actions of multiple enzymes within replisomes. Processivity clamps tether many of these enzymes to DNA, allowing access to the primer/template junction. Many clamp-interacting proteins (CLIPs) are involved in genome maintenance pathways including translesion synthesis (TLS). Despite their abundance, DNA replication in bacteria is not perturbed by these CLIPs. Here we show that while the TLS polymerase Pol IV is largely excluded from moving replisomes, the remodeling of ssDNA binding protein (SSB) upon replisome stalling enriches Pol IV at replication forks. This enrichment is indispensable for Pol IV-mediated TLS on both the leading and lagging strands as it enables Pol IV-processivity clamp binding by overcoming the gatekeeping role of the Pol III epsilon subunit. As we have demonstrated for the Pol IV-SSB interaction, we propose that the binding of CLIPs to the processivity clamp must be preceded by interactions with factors that serve as localization markers for their site of action.

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Role of Tyr-22 in the Binding of Pf3 ssDNA Binding Protein to Nucleic Acids

Biochemistry ◽

10.1021/bi00016a039 ◽

1995 ◽

Vol 34 (16) ◽

pp. 5635-5643 ◽

Cited By ~ 4

Author(s):

Michael D. Powell ◽

Donald M. Gray

Keyword(s):

Nucleic Acids ◽

Binding Protein ◽

Ssdna Binding ◽

Ssdna Binding Protein

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Replication protein A binds RNA and promotes R-loop formation

10.1101/2020.02.11.943977 ◽

2020 ◽

Author(s):

Olga M. Mazina ◽

Srinivas Somarowthu ◽

Lyudmila Y. Kadyrova ◽

Andrey G. Baranovskiy ◽

Tahir H. Tahirov ◽

...

Keyword(s):

Dna Replication ◽

Protein A ◽

Replication Protein A ◽

Replication Protein ◽

Loop Formation ◽

Genome Maintenance ◽

Ssdna Binding ◽

Replication Restart ◽

Ssdna Binding Protein

SUMMARYReplication protein A (RPA), a major eukaryotic ssDNA-binding protein, is essential for all metabolic processes that involve ssDNA including DNA replication, repair, and damage signaling. Surprisingly, we found here that RPA binds RNA in vitro with high affinity. Using native RIP method, we isolated RNA-RPA complexes from human cells. Furthermore, RPA promotes R-loop formation between RNA and homologous dsDNA. R-loops, the three-stranded nucleic acid structure consisting of an RNA-DNA hybrid and the displaced ssDNA strand, are common in human genome. R-loops may play an important role in transcription-coupled homologous recombination and DNA replication restart. We reconstituted the process of replication restart in vitro using RPA-generated R-loops and human DNA polymerases. These findings indicate that RPA may play a role in RNA metabolism and suggest a mechanism of genome maintenance that depends on RPA and RNA.

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Using microsecond single-molecule FRET to determine the assembly pathways of T4 ssDNA binding protein onto model DNA replication forks

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1619819114 ◽

2017 ◽

Vol 114 (18) ◽

pp. E3612-E3621 ◽

Cited By ~ 6

Author(s):

Carey Phelps ◽

Brett Israels ◽

Davis Jose ◽

Morgan C. Marsh ◽

Peter H. von Hippel ◽

...

Keyword(s):

Dna Replication ◽

Single Molecule ◽

Bound States ◽

Function Analysis ◽

Binding Protein ◽

Critical Role ◽

Replication Forks ◽

Ssdna Binding ◽

Single Molecule Fret ◽

Ssdna Binding Protein

DNA replication is a core biological process that occurs in prokaryotic cells at high speeds (∼1 nucleotide residue added per millisecond) and with high fidelity (fewer than one misincorporation event per 107 nucleotide additions). The ssDNA binding protein [gene product 32 (gp32)] of the T4 bacteriophage is a central integrating component of the replication complex that must continuously bind to and unbind from transiently exposed template strands during DNA synthesis. We here report microsecond single-molecule FRET (smFRET) measurements on Cy3/Cy5-labeled primer-template (p/t) DNA constructs in the presence of gp32. These measurements probe the distance between Cy3/Cy5 fluorophores that label the ends of a short (15-nt) segment of ssDNA attached to a model p/t DNA construct and permit us to track the stochastic interconversion between various protein bound and unbound states. The length of the 15-nt ssDNA lattice is sufficient to accommodate up to two cooperatively bound gp32 proteins in either of two positions. We apply a unique multipoint time correlation function analysis to the microsecond-resolved smFRET data obtained to determine and compare the kinetics of various possible reaction pathways for the assembly of cooperatively bound gp32 protein onto ssDNA sequences located at the replication fork. The results of our analysis reveal the presence and translocation mechanisms of short-lived intermediate bound states that are likely to play a critical role in the assembly mechanisms of ssDNA binding proteins at replication forks and other ss duplex junctions.

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