Regulation of DNA Replication through Natural Impediments in the Eukaryotic Genome

Abstract

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.

Keywords: DNA  replication; difficult‐to‐replicate; replication  fork; replication  machinery; replisome; DNA damage; genomic  instability; natural impediments; repetitive DNA; secondary structures.

Figure 1

Telomeres are difficult to replicate. The telomere features including heterochromatin, shelterin proteins, and repeat DNA can hamper progression of the replication fork. The red and green sections in the telomere represent telomeric and subtelomeric sequences, respectively. Although in vivo T-loop formation has not been confirmed in yeast, this structure has been described to form in order to protect the 3′ end overhang from recognition by the DNA repair machinery.

Figure 2

Replication fork barriers (RFBs) at rDNA loci. (A) Each fission yeast rDNA repeat contains a replication origin, 35S rRNA transcription unit, and polar fork-block sites (Ter1, Ter2, Ter3, and RFP4). Ter1, Ter2, and Ter3 sites block the replication fork in a Swi1–Swi3 dependent manner. Sap1 arrests fork progression at Ter1, while Reb1 halts fork progression at Ter2 and Ter3. Fork blockage at RFP4 appears to be dependent on transcription. (B) In budding yeast, collisions between the replication fork and transcription machinery are prevented by Fob1-mediated polar fork blockage at RFBs. This fork block requires the function of Tof1-Csm3. (C) In mammalian cells, TTF-1, a homolog of fission yeast Reb1, arrests fork progression at multiple Sal boxes located near the 3′ end of the 46S rRNA transcription unit. Unlike the cases in budding and fission yeast, fork progression is blocked from both sides in a manner dependent on Timeless–Tipin.

Figure 3

Centromere structures in S. cerevisiae, S. pombe, and humans. (A) Budding yeast has point centromeres, which are comprised of a 125 bp DNA sequence containing centromere DNA elements I (CDEI), II (CDEII), and III (CDEIII). The CBF3 complex binds to CDEIII and is involved in fork pausing at centromeres. (B) Fission yeast has regional centromeres, which consist of 40 to 100 kb DNA sequences including outer repeats (otr), inner repeats (imr), and the central core centromeric sequence (cnt). Pericentric heterochromatin at outer repeats presents histone H3 lysine 9 di- and tri-methylation and may cause fork pausing. RNAi-mediated silencing pathways are involved in releasing RNA polymerase II to maintain replication fork structure at centromeric regions. (C) Human centromeres contain alpha satellite repeats and recruit histone CENP-A. Other human centromere features include histone H3 lysine 9 di/tri-methylation and H3 lysine 4 mono-methylation.

Figure 4

Replication fork pausing at tRNAs and LTRs. (A) The replication fork stalls at tRNA genes in a manner dependent on Tof1 in budding yeast. This fork stalling may prevent fork collapse due to collision between the replication and transcription machineries. Rrm3 sweepase appears to remove non-nucleosomal protein–DNA complex at the fork to facilitate fork progression at tRNA genes. (B) Polar fork pausing at LTRs is mediated by Sap1 in fission yeast. CENP-B-related proteins maintain fork stability at LTRs.

Figure 5

Fork pausing and termination at the fission yeast mating-type locus. Rtf1 binds to RTS1 to prevent fork progression allowing the fork from the opposite direction to progress through the regions. This fork pauses at MPS1 in order to generate an imprint required for recombination-mediated mating-type switching. Swi1–Swi3 and Lsd1/2 are both involved in fork stalling at RTS1 and MPS1.

Figure 6

G-quadruplex structures form at a variety of genomic regions including telomeres and promoters and prevent fork progression leading to genomic instability.

Figure 7

The replication and transcription machinery share the same template DNA, leading to collisions between the two. Timeless-related proteins may promote fork pausing, while Pif1-related DNA helicase facilitate fork progression through highly transcribed regions. Deregulation of fork maintenance at highly transcribed genes results in TAM and TAR.

Figure 8

R-loops formed at the interface between transcription and replication may induce TAM and TAR. Proteins associated with the replisome may play important roles in minimizing R-loop formations to prevent genomic instability.