Replication fork stalling at natural impediments

Abstract

Accurate and complete replication of the genome in every cell division is a prerequisite of genomic stability. Thus, both prokaryotic and eukaryotic replication forks are extremely precise and robust molecular machines that have evolved to be up to the task. However, it has recently become clear that the replication fork is more of a hurdler than a runner: it must overcome various obstacles present on its way. Such obstacles can be called natural impediments to DNA replication, as opposed to external and genetic factors. Natural impediments to DNA replication are particular DNA binding proteins, unusual secondary structures in DNA, and transcription complexes that occasionally (in eukaryotes) or constantly (in prokaryotes) operate on replicating templates. This review describes the mechanisms and consequences of replication stalling at various natural impediments, with an emphasis on the role of replication stalling in genomic instability.

FIG. 1.

Termination of replication in E. coli and B. subtilis. (A) Organization of replication termini in E. coli (left) and B. subtilis (right). In circular bacterial chromosomes, two replication forks (white arrow, clockwise fork; black arrow, counterclockwise fork) start from the bidirectional origin of replication (oriC). Arrest sites are located at the terminus. In E. coli, TerC, TerB, TerF, TerG, and TerJ arrest the clockwise fork (white squares), and TerA, TerD, TerE, TerI, and TerH arrest the counterclockwise fork (black squares). In B. subtilis, TerI (also called IR I), TerIII, and TerV arrest the clockwise fork (white squares), and TerII (also called IR II), TerIV, and TerVI arrest the counterclockwise fork (black squares). Each Ter site contains core (C) and auxiliary (A) sites. TerI (IR I) is the most frequently used termination site. Checkpoint replication arrest sites in the B. subtilis chromosome are indicated by stars: the black star shows the position of arrest of the counterclockwise fork, and the white star shows the position of arrest of the clockwise fork. The positions of sites in both chromosomes are not to scale. (B) Replication termination proteins: Tus of E. coli (left) and RTP of B. subtilis (right) bound to the corresponding Ter sites. In the case of Tus (which binds Ter as a monomer), three models were proposed to explain the asymmetry of fork blocking. (i) The fork-blocking side of Tus contains protruding α helices (gray star), which protect the DNA binding domain from displacement. (ii) Certain amino acid residues on the fork-blocking side of Tus (white star) make protein-protein contacts with the helicase, blocking it. (iii) When the helicase approaches the Ter-Tus complex from the blocking side, it unwinds a part of the Ter site, exposing a particular cytosine residue (black star), which makes contact with Tus, leading to an increased stability of the Ter-Tus complex. In the case of RTP, the asymmetry of fork blocking comes from the different strengths of binding of the RTP dimers to the core and auxiliary sites. Within one Ter site, each core and auxiliary site binds a dimer of RTP (therefore, each Ter site binds four RTP monomers). If the core site is met by the fork first, the fork will be arrested, but if the auxiliary site is met first, both dimers will be displaced and the fork will proceed.

FIG. 2.

Ribosomal RFBs. (A) Organization of rRNA locus in S. cerevisiae. The locus consists of multiple units of direct repeats, each of which contains a 35S RNA transcription unit (which after processing gives rise to 18S, 5.8S, and 25S rRNAs) transcribed by RNA polymerase I, a 5S RNA gene transcribed by RNA polymerase III, and two nontranscribed spacers. One nontranscribed spacer (NTS2) contains a bidirectional origin of replication (ARS), whereas the other nontranscribed spacer (NTS1) contains an RFB that arrests forks that are about to enter the 35S transcription unit (i.e., forks that move from right to left). (B) Comparison of ribosomal RFBs in budding yeast, mammals, and fission yeast. In budding yeast, the RFB is caused by the Fob1 protein; in mammals, the RFB is caused by the TTF-1 protein, which is also a transcription termination factor for rRNAs. Fission yeast combines both scenarios: RFB1 is caused by Sap1, which, like Fob1, is not involved in termination of rRNA transcription, and RFB2 and RFB3 are caused by Reb1, which, like TTF-1, is a transcription termination factor. Arrows, rRNA transcription units. The fork that moves from right to left is blocked.

FIG. 3.

Asymmetrical replication of EBV. OriP, the latent viral origin of replication, consists of the dyad symmetry element (DS) and the family of repeats (FR). Bidirectional replication starts at the DS, which contains four weak binding sites for EBNA-1, but one of the forks is halted at the FR, which contains 20 strong binding sites for EBNA-1. This effectively converts bidirectional replication to unidirectional replication. OriP is a fraction of the viral genome; the picture is not to scale. EBNA-1, EBV nuclear antigen 1; EBERs (EBER-1 and EBER-2), nontranslated, small latent phase-specific EBV-encoded RNAs, transcribed by RNA polymerase III.

FIG. 4.

Organization of the mating-type locus in S. pombe. The current mating type of a cell is determined by the mat1 locus. The switch occurs by means of transferring the genetic information from one of the silent donor loci, mat2-P or mat3-M, into the active mat1 locus. RTS1 (for “replication termination sequence 1”) blocks the fork that moves from left to right, making sure that replication of the mat1 locus occurs by the fork that moves from right to left. The latter pauses at MPS1 (for “mat1 pause site 1”). This pausing leads to the proper placement of the RNA primer, which is the mark of imprinting that regulates the switch.

FIG. 5.

Schematic representation of the head-on and codirectional collisions between replication and transcription in bacteria. The replication fork (trombone view), which moves from left to right, is on the left (leading- and lagging-strand DNA polymerases are shown as ovals; the DNA helicase DnaB is shown as a hexagon). The RNA polymerase with the nascent transcript is on the right. In the case of head-on collision (A), the RNA polymerase transcribes the lagging-strand template; in the case of codirectional collision (B), the RNA polymerase transcribes the leading-strand template.

FIG. 6.

Transcription regulatory elements are punctuation marks for DNA replication. P, promoter; T, transcription terminator. The replication fork is slowed down by a head-on collision with the transcription initiation complex at the promoter (A) or a codirectional passage through the transcription terminator (B), i.e., when it passes the coding region from either direction.

FIG. 7.

Unusual DNA structures and the types of repeats that form them. Structures that can be formed in double-stranded DNA (left) and the types of repetitive sequences that can form them (right) are shown. R, purines; Y, pyrimidines. Identical repetitive units are in black, and the cDNA strands are in white.

FIG. 8.

Two models of triplex-caused DNA polymerization arrest in vitro. (A) On a single-stranded template, a triplex forms behind the polymerase. The template strand folds back on the newly synthesized strand. (B) On a double-stranded (nicked circular) template, a triplex forms in front of the polymerase. The nontemplate strand folds on the duplex in front when displaced by DNA synthesis. Arrows indicate the direction of DNA synthesis. A triplex is formed within a homopurine/homopyrimidine mirror repeat; the pyrimidine strand is white, and the purine strand is black. Note that a pyrimidine triplex is shown in panel A, whereas a purine triplex is shown in panel B, according to the cited studies (see the text).

FIG. 9.

Replication restart. A schematic representation of pathways that act to restart a replication fork, stalled at a “corrupted” template, is shown. See the text for details. *, reversal and rewinding of the fork are most likely assisted by proteins; DNA supercoiling may also contribute to both processes. **, generally, two models that explain replication restart by homologous recombination exist; they differ in the sequence of events. (i) Formation of a D loop (the short arm of the four-way junction invades the parental duplex downstream of the junction) and reestablishment of the replication fork (i.e., reloading of the polymerases and other fork components and resumption of DNA synthesis) are followed by cleavage. (ii) Cleavage (which releases a full chromosomal arm) is followed by the formation of a D loop and reestablishment of the replication fork.

FIG. 10.

Replication model of trinucleotide repeat expansion. (A) Replication stalling caused by the formation of an unusual structure on the lagging-strand template. (B) Fork reversal (possibly assisted by proteins). The 3′ end of the nascent leading strand is single stranded; thus, it tends to fold into a hairpin-like structure. (C) The fork rewinds back to restart (again, assisted by proteins not shown), but the 3′ end of the nascent leading strand is still structured, potentially leading to the repeat's expansion. Ovals, two DNA polymerases in the replication fork; arrows, direction of DNA synthesis; black, structure-prone strand of the repetitive tract; white, complementary strand.