Slx4 regulates DNA damage checkpoint-dependent phosphorylation of the BRCT domain protein Rtt107/Esc4

RTT107 (ESC4, YHR154W) encodes a BRCA1 C-terminal-domain protein that is important for recovery from DNA damage during S phase. Rtt107 is a substrate of the checkpoint protein kinase Mec1, although the mechanism by which Rtt107 is targeted by Mec1 after checkpoint activation is currently unclear. Slx4, a component of the Slx1-Slx4 structure-specific nuclease, formed a complex with Rtt107. Deletion of SLX4 conferred many of the same DNA-repair defects observed in rtt107delta, including DNA damage sensitivity, prolonged DNA damage checkpoint activation, and increased spontaneous DNA damage. These phenotypes were not shared by the Slx4 binding partner Slx1, suggesting that the functions of the Slx4 and Slx1 proteins in the DNA damage response were not identical. Of particular interest, Slx4, but not Slx1, was required for phosphorylation of Rtt107 by Mec1 in vivo, indicating that Slx4 was a mediator of DNA damage-dependent phosphorylation of the checkpoint effector Rtt107. We propose that Slx4 has roles in the DNA damage response that are distinct from the function of Slx1-Slx4 in maintaining rDNA structure and that Slx4-dependent phosphorylation of Rtt107 by Mec1 is critical for replication restart after alkylation damage.     

Rtt107/Esc4 binds silent chromatin and DNA repair proteins using different BRCT motifs

Background  

By screening a plasmid library for proteins that could cause silencing when targeted to the HMR locus in Saccharomyces cerevisiae, we previously reported the identification of Rtt107/Esc4 based on its ability to establish silent chromatin. In this study we aimed to determine the mechanism of Rtt107/Esc4 targeted silencing and also learn more about its biological functions.     

Slx4 and Rtt107 control checkpoint signalling and DNA resection at double-strand breaks

The DNA damage checkpoint pathway is activated in response to DNA lesions and replication stress to preserve genome integrity. However, hyper-activation of this surveillance system is detrimental to the cell, because it might prevent cell cycle re-start after repair, which may also lead to senescence. Here we show that the scaffold proteins Slx4 and Rtt107 limit checkpoint signalling at a persistent double-strand DNA break (DSB) and at uncapped telomeres. We found that Slx4 is recruited within a few kilobases of an irreparable DSB, through the interaction with Rtt107 and the multi-BRCT domain scaffold Dpb11. In the absence of Slx4 or Rtt107, Rad9 binding near the irreparable DSB is increased, leading to robust checkpoint signalling and slower nucleolytic degradation of the 5′ strand. Importantly, in slx4Δ sae2Δ double mutant cells these phenotypes are exacerbated, causing a severe Rad9-dependent defect in DSB repair. Our study sheds new light on the molecular mechanism that coordinates the processing and repair of DSBs with DNA damage checkpoint signalling, preserving genome integrity.     

Dbf4‐dependent kinase and the Rtt107 scaffold promote Mus81‐Mms4 resolvase activation during mitosis

DNA repair by homologous recombination is under stringent cell cycle control. This includes the last step of the reaction, disentanglement of DNA joint molecules (JMs). Previous work has established that JM resolving nucleases are activated specifically at the onset of mitosis. In case of budding yeast Mus81‐Mms4, this cell cycle stage‐specific activation is known to depend on phosphorylation by CDK and Cdc5 kinases. Here, we show that a third cell cycle kinase, Cdc7‐Dbf4 (DDK), targets Mus81‐Mms4 in conjunction with Cdc5—both kinases bind to as well as phosphorylate Mus81‐Mms4 in an interdependent manner. Moreover, DDK‐mediated phosphorylation of Mms4 is strictly required for Mus81 activation in mitosis, establishing DDK as a novel regulator of homologous recombination. The scaffold protein Rtt107, which binds the Mus81‐Mms4 complex, interacts with Cdc7 and thereby targets DDK and Cdc5 to the complex enabling full Mus81 activation. Therefore, Mus81 activation in mitosis involves at least three cell cycle kinases, CDK, Cdc5 and DDK. Furthermore, tethering of the kinases in a stable complex with Mus81 is critical for efficient JM resolution.     

Role of recombination occurring during DNA replication

Unequal crossing over between direct DNA repeats of sister chromosomes occurs during DNA replication in Escherichia coli. Such exchanges yield tandem duplications and thereby increase the expression of the genes involved. Nonhomologous cohesion of sister chromosomes and unequal crossing over were assumed to take place when the replication fork stops. When the replication forks moves continuously, homologous exchanges between sister chromosomes ensure their postreplication repair.     

Rtt107 is required for recruitment of the SMC5/6 complex to DNA double strand breaks

Genome integrity is maintained by a network of DNA damage response pathways, including checkpoints and DNA repair processes. In Saccharomyces cerevisiae, the BRCT domain-containing protein Rtt107/Esc4 is required for the restart of DNA replication after successful repair of DNA damage and for cellular resistance to DNA-damaging agents. In addition to its well characterized interaction with the endonuclease Slx4, Rtt107 interacts with a number of other DNA repair and recombination proteins. These include the evolutionarily conserved SMC5/6 complex, which is involved in numerous chromosome maintenance activities, such as DNA repair, chromosome segregation, and telomere function. The interaction between Rtt107 and the SMC5/6 complex was mediated through the N-terminal BRCT domains of Rtt107 and the Nse6 subunit of SMC5/6 and was independent of methyl methane sulfonate-induced damage and Slx4. Supporting a shared function in the DNA damage response, Rtt107 was required for recruitment of SMC5/6 to DNA double strand breaks. However, this functional relationship did not extend to other types of DNA lesions such as protein-bound nicks. Interestingly, Rtt107 was phosphorylated when SMC5/6 function was compromised in the absence of DNA-damaging agents, indicating a connection beyond the DNA damage response. Genetic analyses revealed that, although a subset of Rtt107 and SMC5/6 functions was shared, these proteins also contributed independently to maintenance of genome integrity.     

Rtt107 BRCT domains act as a targeting module in the DNA damage response

Cells are constantly exposed to assaults that cause DNA damage, which must be detected and repaired to prevent genome instability. The DNA damage response is mediated by key kinases that activate various signaling pathways. In Saccharomyces cerevisiae, one of these kinases is Mec1, which phosphorylates numerous targets, including H2A and the DNA damage protein Rtt107. In addition to being phosphorylated, Rtt107 contains six BRCA1 C-terminal (BRCT) domains, which typically recognize phospho-peptides. Thus Rtt107 represented an opportunity to study complementary aspects of the phosphorylation cascades within one protein. Here we sought to describe the functional roles of the multiple BRCT domains in Rtt107. Rtt107 BRCT5/6 facilitated recruitment to sites of DNA lesions via its interaction with phosphorylated H2A. Rtt107 BRCT3/4 also contributed to Rtt107 recruitment, but BRCT3/4 was not sufficient for recruitment when BRCT5/6 was absent. Intriguingly, both mutations that affected Rtt107 recruitment also abrogated its phosphorylation. Pointing to its modular nature, replacing Rtt107 BRCT5/6 with the BRCT domains from the checkpoint protein Rad9 was able to sustain Rtt107 function. Although Rtt107 physically interacts with both the endonuclease Slx4 and the DNA replication and repair protein Dpb11, only Slx4 was dependent on Rtt107 for its recruitment to DNA lesions. Fusing Rtt107 BRCT5/6 to Slx4, which presumably allows artificial recruitment of Slx4 to DNA lesions, alleviated some phenotypes of rtt107Δ mutants, indicating the functional importance of Slx4 recruitment. Together this data revealed a key function of the Rtt107 BRCT domains for targeting of both itself and its interaction partners to DNA lesions.     

Connecting replication and recombination

Replication forks frequently break and must be repaired by recombination. A reconstituted reaction now allows the factors that coordinate conversion from a recombination intermediate back to a replication fork to be defined. The PriA protein plays a key role in this control.     

Bacteriophage P2: recombination in the superinfection preprophage state and under replication control by phage P4

Genetic crosses (mixed infection, lytic cycle) with bacteriophage P2 are known to give extremely low recombination frequencies, and these are unaffected by the recA status of the host bacterium. We now show the following: (1) the satellite bacteriophage P4, which interacts with P2 in a number of ways, but is quite different from it in terms of DNA replication and its control, is clearly dependent on the host recA+ function for recombination; (2) a chimeric phage (Lindqvist's P2/P4 Hy19), in which P2 replication early genes have been replaced by those of P4, recombines in a recA+-dependent manner; (3) immunity-sensitive P2 phages, in mixed infections of P2-immune bacteria, and hence blocked in their replication, recombine in a recA+-dependent manner; (4) an analysis of the distribution of exchanges based on a simple model confirms that in mixed infections of sensitive cells (where P2 is actively multiplying) recombinational exchanges tend to be statistically clustered in a segment of the chromosome containing the origin of replication, and also shows that, under conditions in which P2 DNA replication is blocked, the distribution of exchanges correlates well with the physical distances between markers on the P2 DNA.     

The Rtt109 histone acetyltransferase facilitates error-free replication to prevent CAG/CTG repeat contractions

Lysine 56 is acetylated on newly synthesized histone H3 in yeast, Drosophila and mammalian cells. All of the proteins involved in histone H3 lysine 56 (H3K56) acetylation are important for maintaining genome integrity. These include Rtt109, a histone acetyltransferase, responsible for acetylating H3K56, Asf1, a histone H3/H4 chaperone, and Hst3 and Hst4, histone deacetylases which remove the acetyl group from H3K56. Here we demonstrate a new role for Rtt109 and H3K56 acetylation in maintaining repetitive DNA sequences in Saccharomyces cerevisiae. We found that cells lacking RTT109 had a high level of CAG/CTG repeat contractions and a twofold increase in breakage at CAG/CTG repeats. In addition, repeat contractions were significantly increased in cells lacking ASF1 and in an hst3Δhst4Δ double mutant. Because the Rtt107/Rtt101 complex was previously shown to be recruited to stalled replication forks in an Rtt109-dependent manner, we tested whether this complex was involved. However, contractions in rtt109Δ cells were not due to an inability to recruit the Rtt107/Rtt101 complex to repeats, as absence of these proteins had no effect on repeat stability. On the other hand, Dnl4 and Rad51-dependent pathways did play a role in creating some of the repeat contractions in rtt109Δ cells. Our results show that H3K56 acetylation by Rtt109 is important for stabilizing DNA repeats, likely by facilitating proper nucleosome assembly at the replication fork to prevent DNA structure formation and subsequent slippage events or fork breakage.     

RMPs: recombination/replication mediator proteins.

Enzymes for DNA replication and recombination need to gain access to single-stranded DNA (ssDNA) but ssDNA-binding proteins (SSBs) present an obstacle to the formation of enzyme-ssDNA replication and recombination complexes. A specialized class of SSBs, which we designate as recombination/replication mediator proteins (RMPs), promotes enzyme- ssDNA assembly by overcoming SSB inhibition. RMPs exhibit strong conservation of function across divergent species, and display species-specific interactions with SSB and enzymes to neutralize the SSB barrier to enzyme-ssDNA assembly.     

Early intermediates in bacteriophage T4 DNA replication and recombination.

We investigated, by density gradients and subsequent electron microscopy, vegetative T4 DNA after single or multiple infection of Escherichia coli with wild-type T4. Our results can be summarized as follows. (i) After single infection (i.e., when early intermolecular recombination could not occur), most, if not all, T4 DNA molecules initiated the first round of replication with a single loop. (ii) After multiple infection, recombinational intermediates containing label from both parents first appeared as early as 1 min after the onset of replication, long before all parental DNA molecules had finished their first round and before secondary replication was detectable. (iii) At the same time, in multiple infections only, complex, highly branched concatemeric T4 DNA first appeared. (iv) Molecules in which two loops or several branches were arranged in tandem were only found after multiple infections. (v) Secondary loops within primary loops were seen after both single and multiple infections, but they were rare and many appeared off center. Thus, recombination in wild-type T4-infected cells occurred very early, and the generation of multiple tandem loops or branches in vegetative T4 DNA depended on recombination. These results are consistent with the previous finding (A. Luder and G. Mosig, Proc. Natl. Acad. Sci. U.S.A. 79:1101-1105, 1982) that most secondary growing points of T4 are not initiated from origin sequences but from recombinational intermediates. By these and previous results, the various DNA molecules that we observed are most readily explained as intermediates in DNA replication and recombination according to a model proposed earlier to explain various other aspects of T4 DNA metabolism (Mosig et al., p. 277-295, in D. Ray, ed., The Initiation of DNA Replication, Academic Press, Inc., New York, 1981).     

High-frequency intermolecular homologous recombination during herpes simplex virus-mediated plasmid DNA replication

Homologous recombination is a prominent feature of herpes simplex virus (HSV) type 1 DNA replication. This has been demonstrated and traditionally studied in experimental settings where repeated sequences are present or are being introduced into a single molecule for subsequent genome isomerization. In the present study, we have designed a pair of unique HSV amplicon plasmids to examine in detail intermolecular homologous recombination (IM-HR) between these amplicon plasmids during HSV-mediated DNA replication. Our data show that IM-HR occurred at a very high frequency: up to 60% of the amplicon concatemers retrieved from virion particles underwent intermolecular homologous recombination. Such a high frequency of IM-HR required that both plasmids be replicated by HSV-mediated replication, as IM-HR events were not detected when either one or both plasmids were replicated by simian virus 40-mediated DNA replication, even with the presence of HSV infection. In addition, the majority of the homologous recombination events resulted in sequence replacement or targeted gene repair, while the minority resulted in sequence insertion. These findings imply that frequent intermolecular homologous recombination may contribute directly to HSV genome isomerization. In addition, HSV-mediated amplicon replication may be an attractive model for studying intermolecular homologous recombination mechanisms in general in a mammalian system. In this regard, the knowledge obtained from such a study may facilitate the development of better strategies for targeted gene correction for gene therapy purposes.     

Initiation of genetic recombination and recombination-dependent replication

Recombination initiates at double-stranded DNA breaks and at single-stranded DNA gaps. These DNA strand discontinuities can arise from DNA-damaging agents and from normal DNA replication when the DNA polymerase encounters an imperfection in the DNA template or another protein. The machinery of homologous recombination acts at these breaks and gaps to promote the events that result in gene recombination, as well as the reattachment of detached replication arms and the resumption of DNA replication. In Escherichia coli, these events require collaboration (RecA, RecBCD, RecFOR, RecQ, RuvABC and SSB proteins) and DNA replication (PriABC proteins and the DNA polymerases). The initial steps common to these recombination and recombination-dependent replication processes are reviewed.     

DNA recombination: the replication connection.

Chromosomal double-strand breaks (DSBs) arise after exposure to ionizing radiation or enzymatic cleavage, but especially during the process of DNA replication itself. Homologous recombination plays a critical role in repair of such DSBs. There has been significant progress in our understanding of two processes that occur in DSB repair: gene conversion and recombination-dependent DNA replication. Recent evidence suggests that gene conversion and break-induced replication are related processes that both begin with the establishment of a replication fork in which both leading- and lagging-strand synthesis occur. There has also been much progress in characterization of the biochemical roles of recombination proteins that are highly conserved from yeast to humans.     

The control of mtDNA replication during differentiation and development

Background

Mitochondrial DNA (mtDNA) is important for energy production as it encodes some of the key genes of electron transfer chain, where the majority of cellular energy is generated through oxidative phosphorylation (OXPHOS). MtDNA replication is mediated by nuclear DNA-encoded proteins or enzymes, which translocate to the mitochondria, and is strictly regulated throughout development. It starts with approximately 200 copies in each primordial germ cell and these copies undergo expansion and restriction events at various stages of development.

Scope of review

I describe the patterns of mtDNA replication at key stages of development. I explain that it is essential to regulate mtDNA copy number and to establish the mtDNA set point in order that the mature, specialised cell acquires the appropriate numbers of mtDNA copy to generate sufficient adenosine triphosphate (ATP) through OXPHOS to undertake its specialised function. I discuss how these processes are dependent on the controlled expression of the nuclear-encoded mtDNA-specific replication factors and that this can be modulated by mtDNA haplotypes. I discuss how these events are altered by certain assisted reproductive technologies, some of which have been proposed to prevent the transmission of mutant mtDNA and others to overcome infertility. Furthermore, some of these technologies are predisposed to transmitting two or more populations of mtDNA, which can be extremely harmful.

Major conclusions

The failure to regulate mtDNA replication and mtDNA transmission during development is disadvantageous.

General significance

Manipulation of oocytes and embryos can lead to significant implications for the maternal-only transmission of mtDNA.This article is part of a Special Issue entitled Frontiers of mitochondrial research.     

BLM helicase-dependent and -independent roles of 53BP1 during replication stress-mediated homologous recombination

Mutations in BLM helicase cause Bloom syndrome, characterized by predisposition to all forms of cancer. We demonstrate that BLM, signal transducer 53BP1, and RAD51 interact during stalled replication. Interactions between the three proteins have functional consequences. Lack of 53BP1 decreases the cell survival and enhanced chromosomal aberration after replication arrest. 53BP1 exhibits both BLM-dependent and -independent anti-recombinogenic functions in human and mouse cells. Both BLM and 53BP1 abrogate endogenous RAD51 foci formation and disrupt RAD51 polymerization. Consequently, loss of BLM and 53BP1 synergistically enhances stress-dependent homologous recombination. These results provide evidence regarding the cooperation between BLM and 53BP1 during maintenance of genomic integrity.     

Chaperone control of the activity and specificity of the histone H3 acetyltransferase Rtt109

Acetylation of Saccharomyces cerevisiae histone H3 on K56 by the histone acetyltransferase (HAT) Rtt109 is important for repairing replication-associated lesions. Rtt109 purifies from yeast in complex with the histone chaperone Vps75, which stabilizes the HAT in vivo. A whole-genome screen to identify genes whose deletions have synthetic genetic interactions with rtt109Delta suggests Rtt109 has functions in addition to DNA repair. We show that in addition to its known H3-K56 acetylation activity, Rtt109 is also an H3-K9 HAT, and we show that Rtt109 and Gcn5 are the only H3-K9 HATs in vivo. Rtt109's H3-K9 acetylation activity in vitro is enhanced strongly by Vps75. Another histone chaperone, Asf1, and Vps75 are both required for acetylation of lysine 9 on H3 (H3-K9ac) in vivo by Rtt109, whereas H3-K56ac in vivo requires only Asf1. Asf1 also physically interacts with the nuclear Hat1/Hat2/Hif1 complex that acetylates H4-K5 and H4-K12. We suggest Asf1 is capable of assembling into chromatin H3-H4 dimers diacetylated on both H4-K5/12 and H3-K9/56.