Break-induced replication: functions and molecular mechanism

Abstract

Break-induced replication (BIR) is the pathway of homologous recombination (HR) conserved from phages to eukaryotes that serves to repair DNA breaks that have only one end. BIR contributes to the repair of broken replication forks and allows telomere lengthening in the absence of telomerase. Nonallelic BIR may lead to translocations and other chromosomal rearrangements. In addition, BIR initiated at sites of microhomology can generate copy number variations (CNVs) and complex chromosomal changes. The level of mutagenesis associated with DNA synthesis in BIR is significantly higher than during normal replication. These features make BIR a likely pathway to promote bursts of genetic changes that fuel cancer progression and evolution.

Figure 1. Multiple functions of BIR

A. Replication of T4 bacteriophage. (i) Several R-loop structures mediate initial rounds of DNA replication. (ii) End-replication problem of lagging strand, collapsed replication forks or resection leave 3′ overhangs that initiate rounds of recombination mediated replication (RDR). (iii) Strand invasion occurring at homologous sequence present at the other end of the same molecule (this is possible because T4 chromosome is terminally redundant; not shown) or at internal part of co-infected molecule (shown) as T4 DNA is circularly permuted. B. Repair of broken replication forks in bacteria. (i) Replication fork runs into a nick and collapses (ii). (iii) Repair of collapsed fork by BIR. Since there is only one origin of replication (ori) extensive synthesis has to be primed from repaired replication fork to terminator (ter). C. Telomere lengthening by recombination. (i) BIR proceeds between telomeres of different chromosomes. (ii) BIR by rolling circle mechanism with extrachromosomal dsDNA telomeric circles.

Figure 2. Assembly of functional replication forks during BIR

A. Initiation of RDR replication in bacteriophage T4. Formation of a D-loop by strand invasion of ssDNA bound to UvsX (RecA-homolog) and UvsY (mediator protein). Gp59 (helicase loading protein) is recruited to the D-loop coated by gp32. Gp59 recruits replicative helicase, gp41, which promotes unwinding of the parent duplex. Polymerase gp43 (together with sliding clamp (gp45) is recruited to the 3′-end of the invading strand and initiates leading strand synthesis. Recruitment of primase gp61 may initiate lagging strand synthesis (not shown). B. Initiation of RDR in E. coli. RecA-mediated strand invasion leads to the formation of a D-loop covered with SSB. PriA recognizes and binds a three-way junction formed by a D-loop and the 3′-end of the invading strand. PriA binding initiates formation of PriA-PriB-DnaT-D-loop complex, which recruits replicative helicase DnaB to the D-loop. DnaB then recruits primase DnaG. Next, Polymerase III is loaded to initiate leading and lagging strand synthesis. C. Initiation of BIR in yeast. Rad51-mediated strand invasion leads to the formation of a D-loop. Initiation of BIR involves the main replicative helicase MCM2-7(shown) and the majority of other proteins required for initiation of S-phase (not shown). The blue hexagon with a question mark represents an unknown hypothetical protein that initiates recruitment of MCMs to the D-loop (similar to PriA and gp59). DNA synthesis associated with BIR is carried out by Polδ, while the exact role of Polε remains unclear.

Figure 3. Models for BIR and associated mutagenesis

A. Single end invasion of 3′-end leads to the formation of a Holliday junction (HJ). Resolution of HJ close to the position of strand invasion leads to the establishment of a unidirectional replication fork, which carries out semi-conservative DNA synthesis. BIR proceeding in this mode is not expected to be mutagenic. B HJ remains unresolved, and BIR proceeds via D-loop migration associated with synchronous synthesis of the leading and lagging strands. Both newly synthesized strands are displaced from their templates, which leads to their conservative inheritance. C. Similar to shown in B, BIR proceeds via D-loop migration associated with conservative inheritance of newly synthesized strands. However, leading strand is synthesized first, while lagging strand synthesis is delayed and proceeds using the leading strand as a template after its displacement. Replication error (for example, nucleotide mis-incorporation; one star) remains uncorrected due to a quick dissociation of newly synthesized DNA from its template (blue oval) (B,C). Replication error (nucleotide mis-incorporation) is present in both strands of DNA (two stars) due to copying of error (C) or due to inability of MMR to discriminate between two newly synthesized strands (B, black oval).

Figure 4. BIR-induced chromosomal rearrangements

A scheme indicating proposed pathways of BIR-induced instability. A. Invasion of 3′-ssDNA into homologous DNA molecule. B. Replication via migrating bubble associated with conservative inheritance of newly synthesized strands (C). D. Premature onset of mitosis due to checkpoint failure interfering with completion of BIR. E. A pause during BIR replication (indicated by “STOP” sign). D and E can lead to one of the following: (i) F. a switch to MMBIR; (ii) G. dissociation of a newly synthesized strand; (iii) H. HJ resolution leading to a half-crossover. I. Translocation resulting from recombination with homologous sequences at ectopic position. J. Half-crossover inducing half-crossover cascades (HCC) where template gets broken and itself initiates second round of recombination. Here broken template gets stabilized by recombination with homologous sequences at ectopic position.