Anatomy and dynamics of DNA replication fork movement in yeast telomeric regions

Replication initiation and replication fork movement in the subtelomeric and telomeric DNA of native Y′ telomeres of yeast were analyzed using two-dimensional gel electrophoresis techniques. Replication origins (ARSs) at internal Y′ elements were found to fire in early-mid-S phase, while ARSs at the terminal Y′ elements were confirmed to fire late. An unfired Y′ ARS, an inserted foreign (bacterial) sequence, and, as previously reported, telomeric DNA each were shown to impose a replication fork pause, and pausing is relieved by the Rrm3p helicase. The pause at telomeric sequence TG(1-3) repeats was stronger at the terminal tract than at the internal TG(1-3) sequences located between tandem Y′ elements. We show that the telomeric replication fork pause associated with the terminal TG(1-3) tracts begins ∼100 bp upstream of the telomeric repeat tract sequence. Telomeric pause strength was dependent upon telomere length per se and did not require the presence of a variety of factors implicated in telomere metabolism and/or known to cause telomere shortening. The telomeric replication fork pause was specific to yeast telomeric sequence and was independent of the Sir and Rif proteins, major known components of yeast telomeric heterochromatin

Recycling of protein subunits during DNA translocation and cleavage by Type I restriction-modification enzymes

The Type I restriction-modification enzymes comprise three protein subunits; HsdS and HsdM that form a methyltransferase (MTase) and HsdR that associates with the MTase and catalyses Adenosine-5′-triphosphate (ATP)-dependent DNA translocation and cleavage. Here, we examine whether the MTase and HsdR components can ‘turnover’ in vitro, i.e. whether they can catalyse translocation and cleavage events on one DNA molecule, dissociate and then re-bind a second DNA molecule. Translocation termination by both EcoKI and EcoR124I leads to HsdR dissociation from linear DNA but not from circular DNA. Following DNA cleavage, the HsdR subunits appear unable to dissociate even though the DNA is linear, suggesting a tight interaction with the cleaved product. The MTases of EcoKI and EcoAI can dissociate from DNA following either translocation or cleavage and can initiate reactions on new DNA molecules as long as free HsdR molecules are available. In contrast, the MTase of EcoR124I does not turnover and additional cleavage of circular DNA is not observed by inclusion of RecBCD, a helicase–nuclease that degrades the linear DNA product resulting from Type I cleavage. Roles for Type I restriction endonuclease subunit dynamics in restriction alleviation in the cell are discussed

DNA damage signalling prevents deleterious telomere addition at DNA breaks

The response to DNA damage involves regulation of multiple essential processes to maximize the accuracy of DNA damage repair and cell survival 1. Telomerase has the potential to interfere with repair by inappropriately adding telomeres to DNA breaks. It was unknown whether cells modulate telomerase in response to DNA damage, to increase the accuracy of repair. Here we report that telomerase action is regulated as a part of the cellular response to a DNA double-strand break (DSB). Using yeast, we show that the major ATR/Mec1 DNA damage signalling pathway regulates telomerase action at DSBs. Upon DNA damage, MEC1-RAD53-DUN1-dependent phosphorylation of the telomerase inhibitor Pif1 occurs. Utilizing a separation of function PIF1 mutation, we show that this phosphorylation is required for the Pif1-mediated telomerase inhibition that takes place specifically at DNA breaks, but not telomeres. Hence DNA damage signalling down-modulates telomerase action at a DNA break via Pif1 phosphorylation, thus preventing aberrant healing of broken DNA ends by telomerase. These findings uncover a novel regulatory mechanism that coordinates competing DNA end-processing activities and thereby promotes DNA repair accuracy and genome integrity

Strong physical constraints on sequence-specific target location by proteins on DNA molecules

Sequence-specific binding to DNA in the presence of competing non-sequence-specific ligands is a problem faced by proteins in all organisms. It is akin to the problem of parking a truck at a loading bay by the side of a road in the presence of cars parked at random along the road. Cars even partially covering the loading bay prevent correct parking of the truck. Similarly on DNA, non-specific ligands interfere with the binding and function of sequence-specific proteins. We derive a formula for the probability that the loading bay is free from parked cars. The probability depends on the size of the loading bay and allows an estimation of the size of the footprint on the DNA of the sequence-specific protein by assaying protein binding or function in the presence of increasing concentrations of non-specific ligand. Assaying for function gives an ‘activity footprint’; the minimum length of DNA required for function rather than the more commonly measured physical footprint. Assaying the complex type I restriction enzyme, EcoKI, gives an activity footprint of ∼66 bp for ATP hydrolysis and 300 bp for the DNA cleavage function which is intimately linked with translocation of DNA by EcoKI. Furthermore, considering the coverage of chromosomal DNA by proteins in vivo, our theory shows that the search for a specific DNA sequence is very difficult; most sites are obscured by parked cars. This effectively rules out any significant role in target location for mechanisms invoking one-dimensional, linear diffusion along DNA

Unloading of homologous recombination factors is required for restoring double-stranded DNA at damage repair loci

Cells use homology‐dependent DNA repair to mend chromosome breaks and restore broken replication forks, thereby ensuring genome stability and cell survival. DNA break repair via homology‐based mechanisms involves nuclease‐dependent DNA end resection, which generates long tracts of single‐stranded DNA required for checkpoint activation and loading of homologous recombination proteins Rad52/51/55/57. While recruitment of the homologous recombination machinery is well characterized, it is not known how its presence at repair loci is coordinated with downstream re‐synthesis of resected DNA. We show that Rad51 inhibits recruitment of proliferating cell nuclear antigen (PCNA), the platform for assembly of the DNA replication machinery, and that unloading of Rad51 by Srs2 helicase is required for efficient PCNA loading and restoration of resected DNA. As a result, srs2Δ mutants are deficient in DNA repair correlating with extensive DNA processing, but this defect in srs2Δ mutants can be suppressed by inactivation of the resection nuclease Exo1. We propose a model in which during re‐synthesis of resected DNA, the replication machinery must catch up with the preceding processing nucleases, in order to close the single‐stranded gap and terminate further resection

Resolution of Joint Molecules by RuvABC and RecG Following Cleavage of the Escherichia coli Chromosome by EcoKI

DNA double-strand breaks can be repaired by homologous recombination involving the formation and resolution of Holliday junctions. In Escherichia coli, the RuvABC resolvasome and the RecG branch-migration enzyme have been proposed to act in alternative pathways for the resolution of Holliday junctions. Here, we have studied the requirements for RuvABC and RecG in DNA double-strand break repair after cleavage of the E. coli chromosome by the EcoKI restriction enzyme. We show an asymmetry in the ability of RuvABC and RecG to deal with joint molecules in vivo. We detect linear DNA products compatible with the cleavage-ligation of Holliday junctions by the RuvABC pathway but not by the RecG pathway. Nevertheless we show that the XerCD-mediated pathway of chromosome dimer resolution is required for survival regardless of whether the RuvABC or the RecG pathway is active, suggesting that crossing-over is a common outcome irrespective of the pathway utilised. This poses a problem. How can cells resolve joint molecules, such as Holliday junctions, to generate crossover products without cleavage-ligation? We suggest that the mechanism of bacterial DNA replication provides an answer to this question and that RecG can facilitate replication through Holliday junctions

Aneuploidy as a mechanism of adaptation to telomerase insufficiency

Cells’ survival is determined by their ability to adapt to constantly changing environment. Adaptation responses involve global changes in transcription, translation, and posttranslational modifications of proteins. In recent years, karyotype changes in adapting populations of single cell organisms have been reported in a number of studies. More recently, we have described aneuploidy as an adaptation mechanism used by populations of budding yeast Saccharomyces cerevisiae to survive telomerase insufficiency induced by elevated growth temperature. Genetic evidence suggests that telomerase insufficiency is caused by decreased levels of the telomerase catalytic subunit Est2. Here, we present experiments arguing that the underlying cause of this phenomenon may be within the telomerase RNA TLC1: changes in the expression of TLC1 as well as mutations in the TLC1 template region affect telomere length equilibrium and the temperature threshold for the induction of telomerase insufficiency. We discuss what lies at the root of telomerase insufficiency, how cell populations overcome it through aneuploidy and whether reversible aneuploidy could be an adaptation mechanism for a variety of environmental stresses