Dynamic elements of replication protein A at the crossroads of DNA replication,recombination, and repair

Abstract

The heterotrimeric eukaryotic Replication protein A (RPA) is a master regulator of numerous DNA metabolic processes. For a long time, it has been viewed as an inert protector of ssDNA and a platform for assembly of various genome maintenance and signaling machines. Later, the modular organization of the RPA DNA binding domains suggested a possibility for dynamic interaction with ssDNA. This modular organization has inspired several models for the RPA-ssDNA interaction that aimed to explain how RPA, the high-affinity ssDNA binding protein, is replaced by the downstream players in DNA replication, recombination, and repair that bind ssDNA with much lower affinity. Recent studies, and in particular single-molecule observations of RPA-ssDNA interactions, led to the development of a new model for the ssDNA handoff from RPA to a specific downstream factor where not only stability and structural rearrangements but also RPA conformational dynamics guide the ssDNA handoff. Here we will review the current knowledge of the RPA structure, its dynamic interaction with ssDNA, and how RPA conformational dynamics may be influenced by posttranslational modification and proteins that interact with RPA, as well as how RPA dynamics may be harnessed in cellular decision making.     

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.     

Expansion of a chromosomal repeat in Escherichia coli: roles of replication, repair, and recombination functions

Background  

Previous studies of gene amplification in Escherichia coli have suggested that it occurs in two steps: duplication and expansion. Expansion is thought to result from homologous recombination between the repeated segments created by duplication. To explore the mechanism of expansion, a 7 kbp duplication in the chromosome containing a leaky mutant version of the lac operon was constructed, and its expansion into an amplified array was studied.     

Spontaneous mutagenesis: the roles of DNA repair, replication, and recombination

There appears to be no dearth of mechanisms to explain spontaneous mutagenesis. In the case of base substitutions, data for bacteriophage T4 and especially for E. coli and S. cerevisiae suggest important roles in spontaneous mutagenesis for the error-prone repair of DNA damage (to produce mutations) and for error-free repair of DNA damage (to avoid mutagenesis). Data from the very limited number of studies on the subject suggest that about 50% of the spontaneous base substitutions in E. coli, and perhaps 90% in S. cerevisiae are due to error-prone DNA repair. On the other hand, spontaneous frameshifts and deletions seem to result from mechanisms involving recombination and replication. Spontaneous insertions have been shown to be important in the strongly polar inactivation of certain loci, but it is less important at other loci. Perhaps with continued study, the term "spontaneous mutagenesis" will be replaced by more specific terms such as 5-methylcytosine deamination mutagenesis, fatty acid oxidation mutagenesis, phenylalanine mutagenesis, and imprecise-recombination mutagenesis. While most studies have concentrated on mutator mutations, the most conclusive data for the actual source of spontaneous mutations have come from the study of antimutator mutations. Further study in this area, perhaps along with an understanding of chemical antimutagens, should be invaluable in clarifying the bases of spontaneous mutagenesis.     

Somatic hypermutation and the three R's: repair, replication and recombination

Somatic hypermutation introduces single base changes into the rearranged variable (V) regions of antigen activated B cells at a rate of approximately 1 mutation per kilobase per generation. This is nearly a million-fold higher than the typical mutation rate in a mammalian somatic cell. Rampant mutation at this level could have a devastating effect, but somatic hypermutation is accurately targeted and tightly regulated. Here, we provide an overview of immunoglobulin gene somatic hypermutation; discuss mechanisms of mutation in model organisms that may be relevant to the hypermutation mechanism; and review recent advances toward understanding the possible role(s) of DNA repair, replication, and recombination in this fascinating process.     

Disease-associated repeat instability and mismatch repair

Expanded tandem repeat sequences in DNA are associated with at least 40 human genetic neurological, neurodegenerative, and neuromuscular diseases. Repeat expansion can occur during parent-to-offspring transmission, and arise at variable rates in specific tissues throughout the life of an affected individual. Since the ongoing somatic repeat expansions can affect disease age-of-onset, severity, and progression, targeting somatic expansion holds potential as a therapeutic target. Thus, understanding the factors that regulate this mutation is crucial. DNA repair, in particular mismatch repair (MMR), is the major driving force of disease-associated repeat expansions. In contrast to its anti-mutagenic roles, mammalian MMR curiously drives the expansion mutations of disease-associated (CAG)·(CTG) repeats. Recent advances have broadened our knowledge of both the MMR proteins involved in disease repeat expansions, including: MSH2, MSH3, MSH6, MLH1, PMS2, and MLH3, as well as the types of repeats affected by MMR, now including: (CAG)·(CTG), (CGG)·(CCG), and (GAA)·(TTC) repeats. Mutagenic slipped-DNA structures have been detected in patient tissues, and the size of the slip-out and their junction conformation can determine the involvement of MMR. Furthermore, the formation of other unusual DNA and R-loop structures is proposed to play a key role in MMR-mediated instability. A complex correlation is emerging between tissues showing varying amounts of repeat instability and MMR expression levels. Notably, naturally occurring polymorphic variants of DNA repair genes can have dramatic effects upon the levels of repeat instability, which may explain the variation in disease age-of-onset, progression and severity. An increasing grasp of these factors holds prognostic and therapeutic potential.     

Paradigms for the three rs: DNA replication, recombination, and repair

The recent decade has engendered a convergence of the otherwise distinct fields of DNA replication, recombination, and repair, as we are learning how these essential transactions can operate in coordination to achieve genomic stability and to ensure cellular viability. In the next decade, we can anticipate a functional understanding of the roles of posttranslational protein modifications in the regulation and prioritizing of pathways for genomic maintenance. The fundamental knowledge gained should lead to more effective clinical intervention in human disease.     

Links between replication, recombination and genome instability in eukaryotes

Double-strand breaks in DNA can be repaired by homologous recombination including break-induced replication. In this reaction, the end of a broken DNA invades an intact chromosome and primes DNA replication resulting in the synthesis of an intact chromosome. Break-induced replication has also been suggested to cause different types of genome rearrangements.     

Genome-wide demethylation promotes triplet repeat instability independently of homologous recombination

Trinucleotide repeat instability is intrinsic to a family of human neurodegenerative diseases. The mechanism leading to repeat length variation is unclear. We previously showed that treatment with the demethylating agent 5-aza-2'-deoxycytidine (5-aza-CdR) dramatically increases triplet repeat instability in mammalian cells. Based on previous reports that demethylation increases homologous recombination (HR), and our own observations that HR destabilizes triplet repeats, we hypothesized that demethylation alters repeat stability by stimulating HR. Here, we test that hypothesis at the adenosine phosphoribosyl transferase (Aprt) locus in CHO cells, where CpG demethylation and HR have both been shown to increase CAG repeat instability. We find that the rate of HR at the Aprt locus is not altered by demethylation. The spectrum of recombinants, however, was shifted from the usual 6:1 ratio of conversions to crossovers to more equal proportions in 5-aza-CdR-treated cells. The subtle influences of demethylation on HR at the Aprt locus are not sufficient to account for its dramatic effects on repeat instability. We conclude that 5-aza-CdR promotes triplet repeat instability independently of HR.     

Instability of the fragile X syndrome repeat in mice: the effect of age, diet and mutations in genes that affect DNA replication, recombination and repair proficiency

Repeat expansion diseases such as fragile X syndrome (FXS) result from increases in the size of a specific tandem repeat array. In addition to large expansions, small changes in repeat number and deletions are frequently seen in FXS pedigrees. No mouse model accurately recapitulates all aspects of this instability, particularly the occurrence of large expansions. This may be due to differences between mice and humans in CIS and/or TRANS-acting factors that affect repeat stability. The identification of such factors may help reveal the expansion mechanism and allow the development of suitable animal models for these disorders. We have examined the effect of age, dietary folate, and mutations in the Werner's syndrome helicase (WRN) and TRP53 genes on FXS repeat instability in mice. WRN facilitates replication of the FXS repeat and enhances Okazaki fragment processing, thereby reducing the incidence of processes that have been suggested to lead to expansion. p53 is a protein involved in DNA damage surveillance and repair. We find two types of repeat instability in these mice, small changes in repeat number that are seen at frequencies approaching 100%, and large deletions which occur at a frequency of about 10%. The frequency of these events was independent of WRN, p53, parental age, or folate levels. The large deletions occur at the same frequency in mice homozygous and heterozygous for the repeat suggesting that they are not the result of an interallelic recombination event. In addition, no evidence of large expansions was seen. Our data thus show that the absence of repeat expansions in mice is not due to a more efficient WRN protein or p53-mediated error correction mechanism, and suggest that these proteins, or the pathways in which they are active, may not be involved in expansion in humans either. Moreover, the fact that contractions occur in the absence of expansions suggests that these processes occur by different mechanisms.     

Comparative genomics of hemiascomycete yeasts: genes involved in DNA replication, repair, and recombination

Among genes conserved from bacteria to mammals are those involved in replicating and repairing DNA. Following the complete sequencing of four hemiascomycetous yeast species during the course of the Genolevures 2 project, we have studied the conservation of 106 genes involved in replication, repair, and recombination in Candida glabrata, Kluyveromyces lactis, Debaryomyces hansenii, and Yarrowia lipolytica and compared them with their Saccharomyces cerevisiae orthologues. We found that proteins belonging to the replication fork and to the nucleotide excision repair pathway were-on the average-more conserved than proteins involved in the checkpoint response to DNA damage or in meiotic recombination. The meiotic recombination proteins Spo11p and Mre11p-Rad50p, involved in making meiotic double-strand breaks (DSBs), are conserved as is Mus81p, involved in resolving meiotic recombination intermediates. Interestingly, genes found in organisms in which DSB-repair is required for proper synapsis during meiosis are also found in C. glabrata, K. lactis, and D. hansenii but not in Y. lipolytica, suggesting that two modes of meiotic recombination have been selected during evolution of the hemiascomycetous yeasts. In addition, we found that SGS1 and TOP1, respectively, a DEAD/DEAH helicase and a type I topoisomerase, are duplicated in C. glabrata and that SRS2, a helicase involved in homologous recombination, is tandemly duplicated in K. lactis. Phylogenetic analyses show that the duplicated SGS1 gene evolved faster than the original gene, probably leading to a specialization of function of the duplicated copy.     

Base excision repair of oxidative DNA damage coupled with removal of a CAG repeat hairpin attenuates trinucleotide repeat expansion

Trinucleotide repeat (TNR) expansion is responsible for numerous human neurodegenerative diseases. However, the underlying mechanisms remain unclear. Recent studies have shown that DNA base excision repair (BER) can mediate TNR expansion and deletion by removing base lesions in different locations of a TNR tract, indicating that BER can promote or prevent TNR expansion in a damage location–dependent manner. In this study, we provide the first evidence that the repair of a DNA base lesion located in the loop region of a CAG repeat hairpin can remove the hairpin, attenuating repeat expansion. We found that an 8-oxoguanine located in the loop region of CAG hairpins of varying sizes was removed by OGG1 leaving an abasic site that was subsequently 5′-incised by AP endonuclease 1, introducing a single-strand breakage in the hairpin loop. This converted the hairpin into a double-flap intermediate with a 5′- and 3′-flap that was cleaved by flap endonuclease 1 and a 3′-5′ endonuclease Mus81/Eme1, resulting in complete or partial removal of the CAG hairpin. This further resulted in prevention and attenuation of repeat expansion. Our results demonstrate that TNR expansion can be prevented via BER in hairpin loops that is coupled with the removal of TNR hairpins.