Regulation of DNA Replication through Natural Impediments in the Eukaryotic Genome

All living organisms need to duplicate their genetic information while protecting it from unwanted mutations, which can lead to genetic disorders and cancer development. Inaccuracies during DNA replication are the major cause of genomic instability, as replication forks are prone to stalling and collapse, resulting in DNA damage. The presence of exogenous DNA damaging agents as well as endogenous difficult‐to‐replicate DNA regions containing DNA–protein complexes, repetitive DNA, secondary DNA structures, or transcribing RNA polymerases, increases the risk of genomic instability and thus threatens cell survival. Therefore, understanding the cellular mechanisms required to preserve the genetic information during S phase is of paramount importance. In this review, we will discuss our current understanding of how cells cope with these natural impediments in order to prevent DNA damage and genomic instability during DNA replication

Lingering single-strand breaks trigger Rad51-independent homology-directed repair of collapsed replication forks in the polynucleotide kinase/phosphatase mutant of fission yeast

<div><p>The DNA repair enzyme polynucleotide kinase/phosphatase (PNKP) protects genome integrity by restoring ligatable 5’-phosphate and 3’-hydroxyl termini at single-strand breaks (SSBs). In humans, PNKP mutations underlie the neurological disease known as MCSZ, but these individuals are not predisposed for cancer, implying effective alternative repair pathways in dividing cells. Homology-directed repair (HDR) of collapsed replication forks was proposed to repair SSBs in PNKP-deficient cells, but the critical HDR protein Rad51 is not required in PNKP-null (<i>pnk1Δ</i>) cells of <i>Schizosaccharomyces pombe</i>. Here, we report that <i>pnk1Δ</i> cells have enhanced requirements for Rad3 (ATR/Mec1) and Chk1 checkpoint kinases, and the multi-BRCT domain protein Brc1 that binds phospho-histone H2A (γH2A) at damaged replication forks. The viability of <i>pnk1Δ</i> cells depends on Mre11 and Ctp1 (CtIP/Sae2) double-strand break (DSB) resection proteins, Rad52 DNA strand annealing protein, Mus81-Eme1 Holliday junction resolvase, and Rqh1 (BLM/WRN/Sgs1) DNA helicase. Coupled with increased sister chromatid recombination and Rad52 repair foci in <i>pnk1Δ</i> cells, these findings indicate that lingering SSBs in <i>pnk1Δ</i> cells trigger Rad51-independent homology-directed repair of collapsed replication forks. From these data, we propose models for HDR-mediated tolerance of persistent SSBs with 3’ phosphate in <i>pnk1Δ</i> cells.</p></div

Brc1 binding to γH2A is important in <i>pnk1Δ</i> cells.

<p>Tenfold serial dilutions of cells were exposed to the indicated DNA damaging agents. Plates were incubated at 30°C for 3 to 4 days. Doubling times were determined with cells grown in liquid YES media at 32°C. Note that the <i>brc1-T672A</i> and <i>brc1-K710M</i> alleles contain a C-terminal 2GFP tag, which under some conditions can be observed to partially impair Brc1, thus strains with these alleles should be compared to wild type Brc1 tagged with 2GFP (<i>brc1</i>:<i>2GFP</i>).</p

Increased spontaneous recombination in <i>pnk1Δ</i> cells.

<p><b>A)</b> Schematic of the non-tandem direct repeat of <i>ade6</i>^<i>-</i> heteroalleles used for measuring spontaneous recombinant frequencies. Conversion types events result in Ade⁺ His^- colonies, whereas deletion types events result in Ade⁺ His⁺ colonies. <b>B)</b> Recombination frequencies (per 10⁴ viable cells ± SD) of the following strains: wild type (1.41 ± 0.57), <i>brc1Δ</i> (1.62 ± 1.16), <i>pnk1Δ</i> (4.78 ± 1.62), <i>brc1Δ pnk1Δ</i> (3.91 ± 2.3). Deletion types and conversion types were determined by replica-plating. Error bars correspond to standard deviations of the means. Asterisk depicts statistically significant differences with wild type and + symbol with <i>pnk1Δ</i>, as determined by two-tailed Student T-test, p-value ≤ 0.05.</p

Requirement for Rad52 in <i>pnk1Δ</i> cells.

<p><b>A)</b> A <i>pnk1Δ rad51Δ</i> is viable but it displays increased HU and MMS sensitivity relative to <i>rad51Δ</i>. Tenfold serial dilutions of cells were exposed to the indicated DNA damaging agents. Plates were incubated at 30°C for 3 to 4 days. Doubling times were determined with cells grown in liquid YES media at 32°C. <b>B)</b> Rad52 is crucial for viability in <i>pnk1Δ</i> cells. Strains with <i>pnk1Δ</i> or <i>rad52Δ</i> mutations, or the double mutant, in a <i>ura4-D18</i> background, were transformed with the pRad52 plasmid containing the <i>rad52</i>⁺ gene and <i>ura4</i>⁺ selectable marker. These strains and controls (wild type with <i>ura4</i>⁺ or <i>ura4-D18</i>) were incubated on rich YES plates (no selection for <i>ura4</i>⁺), LAH media (selection for <i>ura4</i>⁺), or 5-FOA plates (counter selection for <i>ura4</i>⁺). Relative to <i>pnk1Δ</i> or <i>rad52Δ</i> single mutants carrying pRad52, the <i>pnk1Δ rad52Δ</i>, double mutant grew very poorly on 5-FOA plates. These results were confirmed by determining through microscopic observation the percentage of cells that can form colonies on 5-FOA plates.</p

Models for replication-coupled repair of SSBs with 3’ phosphate terminus.

<p>1a, replication fork collapses upon encountering a SSB with 3’ phosphate in the lagging strand template. 1b, resection of DSB followed by strand invasion of the sister chromatid using 3’ single-strand overhang containing 3’ phosphate. 1c, resolution of D-loop or Holliday junction by Mus81-Eme1. 1d, replication by converging fork, leaving a single-strand gap with 3’ phosphate. 2a, replication fork collapses upon encountering a SSB with 3’ phosphate in the leading strand template. 2b, converging fork collapses at SSB, leaving DSB with 3’ phosphate terminus. 2c, resection of DNA end with 3’ hydroxyl, followed by strand invasion of the sister chromatid. 2d, completion of repair by SDSA leaves a single-strand gap with 3’ phosphate.</p

Swi10-Rad16 3’ flap endonuclease is not required in <i>pnk1Δ</i> cells.

<p>Tenfold serial dilutions of cells were exposed to the indicated DNA damaging agents. Plates were incubated at 30°C for 3 to 4 days.</p

Rad52 and RPA foci increase in <i>pnk1Δ</i> cells and further increase in <i>brc1Δ pnk1Δ</i> cells.

<p>Cells expressing Rad52-YFP (A) or RPA-GFP (B) were cultured in minimal medium at 25°C until mid-log phase. The larger increase of Rad52 foci relative to RPA foci n <i>pnk1Δ brc1Δ</i> cells might reflect foci duration. Cell cycle phase markers (cell length, number and position of nuclei as visualized by nucleoplasmic Rad52-YFP or RPA-GFP, absence or presence of septum) was used to estimate cell cycle position. The columns indicate, from top to bottom, cell cycle phases of cells with foci: M or G1 (M-G1), G2, S or early G2 (S-G2). Error bars correspond to standard deviation of the mean. Asterisk (*) and plus (+) symbols indicate statistically significant differences with wild type or <i>pnk1Δ</i> strains, respectively, as determine by two-tailed Student T-test, p-value ≤ 0.05.</p

DNA damage checkpoint activation and requirement for checkpoint proteins in <i>pnk1Δ</i> cells.

<p><b>A)</b> Effects of combining <i>pnk1Δ</i> with <i>rad3Δ</i> or <i>brc1Δ</i> mutations. Tenfold serial dilutions of cells were exposed to the indicated DNA damaging agents. Plates were incubated at 30°C for 3 to 4 days. <b>B)</b> Chk1 also undergoes activating phosphorylation in untreated and HU-treated <i>pnk1Δ</i> cells, unlike in wild type. Cells were incubated with 10 mM HU, 5 μM CPT, or 0.01% MMS for 3 hours. <b>C)</b> Effects of combining <i>pnk1Δ</i> with <i>chk1Δ</i> or <i>brc1Δ</i> mutations. <b>D)</b> Effects of combining <i>pnk1Δ</i> with <i>chk1Δ</i> or <i>cds1Δ</i>.</p

Mus81-Eme1 resolvase and Rqh1 DNA helicase are essential in <i>pnk1Δ</i> mutant cells.

<p><b>A)</b> The few viable <i>pnk1Δ mus81Δ</i> viable cells recovered from genetic crosses are very sick compared to single mutants. <b>B)</b> The few viable <i>pnk1Δ eme1Δ</i> viable cells recovered from genetic crosses are very sick compared to single mutants. Tenfold serial dilutions of cells were plated and incubated at 30°C for 3 to 4 days. <b>C)</b> Elimination of Rad51 does not suppress <i>pnk1Δ mus81Δ</i> synthetic lethality. Tetrad analysis of <i>pnk1Δ rad51Δ</i> x <i>mus81Δ</i> cross. <b>D)</b> Elimination of Rad54 does not suppress <i>pnk1Δ mus81Δ</i> synthetic lethality. Tetrad analysis of <i>pnk1Δ rad54Δ</i> x <i>mus81Δ</i> cross. <b>E)</b> Tetrad analysis of mating between <i>pnk1Δ</i> and <i>rqh1Δ</i> strains.</p