DNA ligase 4 stabilizes the ribosomal DNA array upon fork collapse at the replication fork barrier

DNA double-strand breaks (DSB) were shown to occur at the replication fork barrier in the ribosomal DNA of Saccharomyces cerevisiae using 2D-gel electrophoresis. Their origin, nature and magnitude, however, have remained elusive. We quantified these DSBs and show that a surprising 14% of replicating ribosomal DNA molecules are broken at the replication fork barrier in replicating wild-type cells. This translates into an estimated steady-state level of 7–10 DSBs per cell during S-phase. Importantly, breaks detectable in wild-type and sgs1 mutant cells differ from each other in terms of origin and repair. Breaks in wild-type, which were previously reported as DSBs, are likely an artefactual consequence of nicks nearby the rRFB. Sgs1 deficient cells, in which replication fork stability is compromised, reveal a class of DSBs that are detectable only in the presence of functional Dnl4. Under these conditions, Dnl4 also limits the formation of extrachromosomal ribosomal DNA circles. Consistently, dnl4 cells displayed altered fork structures at the replication fork barrier, leading us to propose an as yet unrecognized role for Dnl4 in the maintenance of ribosomal DNA stability.     

Ribosomal DNA replication fork barrier and HOT1 recombination hot spot: shared sequences but independent activities

In the ribosomal DNA of Saccharomyces cerevisiae, sequences in the nontranscribed spacer 3' of the 35S ribosomal RNA gene are important to the polar arrest of replication forks at a site called the replication fork barrier (RFB) and also to the cis-acting, mitotic hyperrecombination site called HOT1. We have found that the RFB and HOT1 activity share some but not all of their essential sequences. Many of the mutations that reduce HOT1 recombination also decrease or eliminate fork arrest at one of two closely spaced RFB sites, RFB1 and RFB2. A simple model for the juxtaposition of RFB and HOT1 sequences is that the breakage of strands in replication forks arrested at RFB stimulates recombination. Contrary to this model, we show here that HOT1-stimulated recombination does not require the arrest of forks at the RFB. Therefore, while HOT1 activity is independent of replication fork arrest, HOT1 and RFB require some common sequences, suggesting the existence of a common trans-acting factor(s).     

The nonmutagenic repair of broken replication forks via recombination

When replication forks stall or collapse at sites of DNA damage, there are two avenues for fork rescue. Mutagenic translesion synthesis by a special class of DNA polymerases can move a fork past the damage, but can leave behind mutations. The alternative nonmutagenic pathways for fork repair involve cellular recombination systems. In bacteria, nonmutagenic repair of replication forks may occur as often as once per cell per generation, and is the favored path for fork restoration under normal growth conditions. Replication fork repair is almost certainly the major function of bacterial recombination systems, and was probably the impetus for the evolution of recombination systems. Increasingly, the nonmutagenic repair of replication forks is seen as a major function of eukaryotic recombination systems as well.     

The replication fork barrier site forms a unique structure with Fob1p and inhibits the replication fork

The replication fork barrier site (RFB) is an approximately 100-bp DNA sequence located near the 3' end of the rRNA genes in the yeast Saccharomyces cerevisiae. The gene FOB1 is required for this RFB activity. FOB1 is also necessary for recombination in the ribosomal DNA (rDNA), including increase and decrease of rDNA repeat copy number, production of extrachromosomal rDNA circles, and possibly homogenization of the repeats. Despite the central role that Foblp plays in both replication fork blocking and rDNA recombination, the molecular mechanism by which Fob1p mediates these activities has not been determined. Here, I show by using chromatin immunoprecipitation, gel shift, footprinting, and atomic force microscopy assays that Fob1p directly binds to the RFB. Fob1p binds to two separated sequences in the RFB. A predicted zinc finger motif in Fob1p was shown to be essential for the RFB binding, replication fork blocking, and rDNA recombination activities. The RFB seems to wrap around Fob1p, and this wrapping structure may be important for function in the rDNA repeats.     

A model of the replication fork blocking protein Fob1p based on the catalytic core domain of retroviral integrases

The replication fork blocks are common in both prokaryotes and eukaryotes. In most cases, these blocks are associated with increased levels of mitotic recombination. One of the best-characterized replication fork blocks in eukaryotes is found in ribosomal DNA (rDNA) repeats of Saccharomyces cerevisiae. It has been shown that the replication fork blocking protein Fob1p regulates the recombination rate and the number of rDNA copies in S. cerevisiae, but the mechanistic aspects of these events are still poorly understood. Sequence profile searches revealed that Fob1p is related to retroviral integrases. Subsequently, the catalytic domain of HIV-1 integrase was used as a template to build a reliable three-dimensional model of Fob1p. Structural insights from this study may be useful in explaining Fob1p-mediated formation of extrachromosomal rDNA circles that accelerate aging in yeast and recombination events that lead to expansion or contraction of rDNA.     

Checkpoint responses to replication fork barriers

The fidelity of DNA replication is of paramount importance to the maintenance of genome integrity. When an active replication fork is perturbed, multiple cellular pathways are recruited to stabilize the replication apparatus and to help to bypass or correct the causative problem. However, if the problem is not corrected, the fork may collapse, exposing free DNA ends to potentially inappropriate processing. In prokaryotes, replication fork collapse promotes the activity of recombination proteins to restore a replication fork. Recent work has demonstrated that recombination is also intimately linked to replication in eukaryotic cells, and that recombination proteins are recruited to collapsed, but not stalled, replication forks. In this review we discuss the different types of potential replication fork barriers (RFB) and how these distinct RFBs can result in different DNA structures at the stalled replication fork. The DNA structure checkpoints which act within S phase respond to different RFBs in different ways and we thus discuss the processes that are controlled by the DNA replication checkpoints, paying particular attention to the function of the intra-S phase checkpoint that stabilises the stalled fork.     

Dna2 helicase/nuclease causes replicative fork stalling and double-strand breaks in the ribosomal DNA of Saccharomyces cerevisiae

We have proposed that faulty processing of arrested replication forks leads to increases in recombination and chromosome instability in Saccharomyces cerevisiae and contributes to the shortened lifespan of dna2 mutants. Now we use the ribosomal DNA locus, which is a good model for all stages of DNA replication, to test this hypothesis. We show directly that DNA replication pausing at the ribosomal DNA replication fork barrier (RFB) is accompanied by the occurrence of double-strand breaks near the RFB. Both pausing and breakage are elevated in the early aging, hypomorphic dna2-2 helicase mutant. Deletion of FOB1, encoding the fork barrier protein, suppresses the elevated pausing and DSB formation, and represses initiation at rDNA ARSs. The dna2-2 mutation is synthetically lethal with deltarrm3, encoding another DNA helicase involved in rDNA replication. It does not appear to be the case that the rDNA is the only determinant of genome stability during the yeast lifespan however since strains carrying deletion of all chromosomal rDNA but with all rDNA supplied on a plasmid, have decreased rather than increased lifespan. We conclude that the replication-associated defects that we can measure in the rDNA are symbolic of similar events occurring either stochastically throughout the genome or at other regions where replication forks move slowly or stall, such as telomeres, centromeres, or replication slow zones.     

Role of recombination and replication fork restart in repeat instability

Eukaryotic genomes contain many repetitive DNA sequences that exhibit size instability. Some repeat elements have the added complication of being able to form secondary structures, such as hairpin loops, slipped DNA, triplex DNA or G-quadruplexes. Especially when repeat sequences are long, these DNA structures can form a significant impediment to DNA replication and repair, leading to DNA nicks, gaps, and breaks. In turn, repair or replication fork restart attempts within the repeat DNA can lead to addition or removal of repeat elements, which can sometimes lead to disease. One important DNA repair mechanism to maintain genomic integrity is recombination. Though early studies dismissed recombination as a mechanism driving repeat expansion and instability, recent results indicate that mitotic recombination is a key pathway operating within repetitive DNA. The action is two-fold: first, it is an important mechanism to repair nicks, gaps, breaks, or stalled forks to prevent chromosome fragility and protect cell health; second, recombination can cause repeat expansions or contractions, which can be deleterious. In this review, we summarize recent developments that illuminate the role of recombination in maintaining genome stability at DNA repeats.     

When replication forks stop

DNA synthesis is an accurate and very processive phenomenon, yet chromosome replication does not proceed at a constant rate and progression of the replication fork can be impeded. Several structural and functional features of the template can modulate the rate of progress of the replication fork. These include DNA secondary structures, DNA damage and occupied protein-binding sites. In addition, prokaryotes contain sites where replication is specifically arrested. DNA regions at which the replication machinery is blocked or transiently slowed could be particularly susceptible to genome rearrangements. Illegitimate recombination, a ubiquitous phenomenon which may have dramatic consequences, occurs by a variety of mechanisms. The observation that some rearrangements might be facilitated by a pause in replication could provide a clue in elucidating these processes. In support of this, some homologous and illegitimate recombination events have already been correlated with replication pauses or arrest sites.     

PriA and phage T4 gp59: factors that promote DNA replication on forked DNA substrates microreview

The initiation of DNA synthesis on forked DNA templates is a vital process in the replication and maintenance of cellular chromosomes. Two proteins that promote replisome assembly on DNA forks have so far been identified. In phage T4 development the gene 59 protein (gp59) assembles replisomes at D-loops, the sites of homologous strand exchange. Bacterial PriA protein plays an analogous function, most probably restarting replication after replication fork arrest with the aid of homologous recombination proteins, and PriA is also required for phage Mu replication by transposition. Gp59 and PriA exhibit similar DNA fork binding activities, but PriA also has a 3' to 5' helicase activity that can promote duplex opening for replisome assembly. The helicase activity allows PriA's repertoire of templates to be more diverse than that of gp59. It may give PriA the versatility to restart DNA replication without recombination on arrested replication forks that lack appropriate duplex openings.     

The Saccharomyces Pif1p DNA helicase and the highly related Rrm3p have opposite effects on replication fork progression in ribosomal DNA

Replication of Saccharomyces ribosomal DNA (rDNA) proceeds bidirectionally from origins in a subset of the approximately 150 tandem repeats, but the leftward-moving fork stops when it encounters the replication fork barrier (RFB). The Pif1p helicase and the highly related Rrm3p were rDNA associated in vivo. Both proteins affected rDNA replication but had opposing effects on fork progression. Pif1p helped maintain the RFB. Rrm3p appears to be the replicative helicase for rDNA as it acted catalytically to promote fork progression throughout the rDNA. Loss of Rrm3p increased rDNA breakage and accumulation of rDNA circles, whereas breakage and circles were less common in pif1 cells. These data support a model in which replication fork pausing causes breakage and recombination in the rDNA.     

Overcoming natural replication barriers: differential helicase requirements

DNA sequences that form secondary structures or bind protein complexes are known barriers to replication and potential inducers of genome instability. In order to determine which helicases facilitate DNA replication across these barriers, we analyzed fork progression through them in wild-type and mutant yeast cells, using 2-dimensional gel-electrophoretic analysis of the replication intermediates. We show that the Srs2 protein facilitates replication of hairpin-forming CGG/CCG repeats and prevents chromosome fragility at the repeat, whereas it does not affect replication of G-quadruplex forming sequences or a protein-bound repeat. Srs2 helicase activity is required for hairpin unwinding and fork progression. Also, the PCNA binding domain of Srs2 is required for its in vivo role of replication through hairpins. In contrast, the absence of Sgs1 or Pif1 helicases did not inhibit replication through structural barriers, though Pif1 did facilitate replication of a telomeric protein barrier. Interestingly, replication through a protein barrier but not a DNA structure barrier was modulated by nucleotide pool levels, illuminating a different mechanism by which cells can regulate fork progression through protein-mediated stall sites. Our analyses reveal fundamental differences in the replication of DNA structural versus protein barriers, with Srs2 helicase activity exclusively required for fork progression through hairpin structures.     

Regulation of DNA replication on subchromosomal units of mammalian cells

The regulation of DNA replication at a subchromosomal level in mammalian cells has been investigated. DNA fiber autoradiographs were prepared from mouse L-929 cells pulse labeled with (3H)thymidine. Initiation events and subsequent chain growth occurring over short stretches (up to three replication units in length) of chromosomal DNA were analyzed. The results show that adjacent units usually initiate replication synchronously and that this synchrony is related to the proximity of initiation sites. In addition, adjacent units are of similar size and the rates of replication fork progression within units and on adjacent units are similar. The rate of fork progression increases with increasing replication unit size. Finally, no evidence for fixed termination sites for the units has been found. These observations suggest that despite large variations in size of replication units, timing of initiation events, and rates of fork progression found in chromosomal DNA as a whole, these processes are closely regulated within subchromosomal clusters of active replication units.