Genome instability is a hallmark of cancer cells, and defective DNA replication, repair and recombination have been linked to its etiology. Increasing evidence suggests that proteins influencing S-phase processes such as replication fork movement and stability, repair events and replication completion, have significant roles in maintaining genome stability. DNA damage and replication stress activate a signal transduction cascade, often referred to as the checkpoint response. A central goal of the replication checkpoint is to maintain the integrity of the replication forks while facilitating replication completion and DNA repair and coordinating these events with cell cycle transitions. Progression through the cell cycle in spite of defective or incomplete DNA synthesis or unrepaired DNA lesions may result in broken chromosomes, genome aberrations, and an accumulation of mutations. In this review we discuss the multiple roles of the replication checkpoint during replication and in response to replication stress, as well as the enzymatic activities that cooperate with the checkpoint pathway to promote fork resumption and repair of DNA lesions thereby contributing to genome integrity.
"Cds1p can maintain the stability of stalled replication forks by regulating Mus81p-Eme1p nuclease, Rqh1p, a RecQ-family helicase involved in suppressing inappropriate recombination during replication, and Rad60, a protein required for recombinational repair during replication. This interaction reveals a direct link between Cds1p kinase and recombinational repair processes (Kai and Wang, 2003; Branzei and Foiani, 2009). Thus in the future it should be tested whether Rgf1p might play some role in processing blocked replication forks to prevent subsequent recombinogenic processes or whether Rgf1p's function in cytoskeleton dynamics might indirectly feed into these Rad52p-dependent processes. "
[Show abstract][Hide abstract] ABSTRACT: Guanine nucleotide exchange factors (GEFs) control many aspects of cell morphogenesis by turning on Rho-GTPases. The fission yeast exchange factor Rgf1p (Rho gef1) specifically regulates Rho1p during polarized growth and localizes to cortical sites. Here we report that Rgf1p is re-localized to the cell nucleus during the stalled replication caused by hydroxyurea (HU). Import to the nucleus is mediated by a nuclear localization sequence (NLS) at the N-terminus of Rgf1p, while release into the cytoplasm requires two leucine-rich nuclear export sequences (NES) at the C-terminus. Moreover, Rgf1p nuclear accumulation during replication arrest depends on the 14-3-3 chaperone Rad24p and on the DNA replication checkpoint kinase Cds1p. Both proteins control the nuclear accumulation of Rgf1p by inhibition of its nuclear export. A mutant, Rgf1p-9A, that substitutes 9 Ser potential phosphorylation Cds1p sites for Ala fails to accumulate in the nucleus in response to replication stress and this correlates with a severe defect in survival in the presence of HU. In conclusion, we propose that the regulation of Rgf1p could be part of the mechanism by which Cds1p and Rad24p promote survival in the presence of chronic replication stress. It will be of general interest to understand whether the same is true for homologs of Rgf1p in budding yeast and higher eukaryotes.
Molecular biology of the cell 01/2014; 25(7). DOI:10.1091/mbc.E13-11-0689 · 4.47 Impact Factor
"Stalled replication forks must be restarted in order to complete replication. Checkpoint related kinases, such as Mec1 and Rad53 (Chk2 ortholog) in yeast, play important roles in regulating restart by activating appropriate pathways to resolve fork blockage (63,64). One important step in this process is to reduce the frequency of HR that could lead to destabilization of replication forks and generate toxic HR intermediates (63,64). "
[Show abstract][Hide abstract] ABSTRACT: Replication Protein A (RPA) is a heterotrimeric protein complex that binds single-stranded DNA. In plants, multiple genes
encode the three RPA subunits (RPA1, RPA2 and RPA3), including five RPA1-like genes in Arabidopsis. Phylogenetic analysis
suggests two distinct groups composed of RPA1A, RPA1C, RPA1E (ACE group) and RPA1B, RPA1D (BD group). ACE-group members are
transcriptionally induced by ionizing radiation, while BD-group members show higher basal transcription and are not induced
by ionizing radiation. Analysis of rpa1 T-DNA insertion mutants demonstrates that although each mutant line is likely null, all mutant lines are viable and display
normal vegetative growth. The rpa1c and rpa1e single mutants however display hypersensitivity to ionizing radiation, and combination of rpa1c and rpa1e results in additive hypersensitivity to a variety of DNA damaging agents. Combination of the partially sterile rpa1a with rpa1c results in complete sterility, incomplete synapsis and meiotic chromosome fragmentation, suggesting an early role for RPA1C
in promoting homologous recombination. Combination of either rpa1c and/or rpa1e with atr revealed additive hypersensitivity phenotypes consistent with each functioning in unique repair pathways. In contrast, rpa1b rpa1d double mutant plants display slow growth and developmental defects under non-damaging conditions. We show these defects in
the rpa1b rpa1d mutant are likely the result of defective DNA replication leading to reduction in cell division.
Nucleic Acids Research 12/2013; DOI:10.1093/nar/gkt1292 · 9.11 Impact Factor
"Whereas the SDSA pathway leads to generation of gene conversion products, resolution of double Holliday junctions can lead to both gene conversions as well as crossovers (17,25). Although HR constitutes an error-free damage tolerance pathway, the intermediates formed during this process are toxic because they can trigger cell-cycle arrest and cell death (26–28), and HR is thus under very tight control (17,29,30). "
[Show abstract][Hide abstract] ABSTRACT: Stalling of replication forks at unrepaired DNA lesions can result in discontinuities opposite the damage in the newly synthesized DNA strand. Translesion synthesis or facilitating the copy from the newly synthesized strand of the sister duplex by template switching can overcome such discontinuities. During template switch, a new primer-template junction has to be formed and two mechanisms, including replication fork reversal and D-loop formation have been suggested. Genetic evidence indicates a major role for yeast Rad5 in template switch and that both Rad5 and its human orthologue, Helicase-like transcription factor (HLTF), a potential tumour suppressor can facilitate replication fork reversal. This study demonstrates the ability of HLTF and Rad5 to form a D-loop without requiring ATP binding and/or hydrolysis. We also show that this strand-pairing activity is independent of RAD51 in vitro and is not mechanistically related to that of another member of the SWI/SNF family, RAD54. In addition, the 3'-end of the invading strand in the D-loop can serve as a primer and is extended by DNA polymerase. Our data indicate that HLTF is involved in a RAD51-independent D-loop branch of template switch pathway that can promote repair of gaps formed during replication of damaged DNA.
Nucleic Acids Research 11/2013; 42(3). DOI:10.1093/nar/gkt1040 · 9.11 Impact Factor
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