RPA activates the XPF‐ERCC1 endonuclease to initiate processing of DNA interstrand crosslinks

During replication‐coupled DNA interstrand crosslink (ICL) repair, the XPF‐ERCC1 endonuclease is required for the incisions that release, or “unhook”, ICLs, but the mechanism of ICL unhooking remains largely unknown. Incisions are triggered when the nascent leading strand of a replication fork strikes the ICL. Here, we report that while purified XPF‐ERCC1 incises simple ICL‐containing model replication fork structures, the presence of a nascent leading strand, modelling the effects of replication arrest, inhibits this activity. Strikingly, the addition of the single‐stranded DNA (ssDNA)‐binding replication protein A (RPA) selectively restores XPF‐ERCC1 endonuclease activity on this structure. The 5′–3′ exonuclease SNM1A can load from the XPF‐ERCC1‐RPA‐induced incisions and digest past the crosslink to quantitatively complete the unhooking reaction. We postulate that these collaborative activities of XPF‐ERCC1, RPA and SNM1A might explain how ICL unhooking is achieved in vivo.     

HOT1 is a mammalian direct telomere repeat‐binding protein contributing to telomerase recruitment

Telomeres are repetitive DNA structures that, together with the shelterin and the CST complex, protect the ends of chromosomes. Telomere shortening is mitigated in stem and cancer cells through the de novo addition of telomeric repeats by telomerase. Telomere elongation requires the delivery of the telomerase complex to telomeres through a not yet fully understood mechanism. Factors promoting telomerase–telomere interaction are expected to directly bind telomeres and physically interact with the telomerase complex. In search for such a factor we carried out a SILAC‐based DNA–protein interaction screen and identified HMBOX1, hereafter referred to as homeobox telomere‐binding protein 1 (HOT1). HOT1 directly and specifically binds double‐stranded telomere repeats, with the in vivo association correlating with binding to actively processed telomeres. Depletion and overexpression experiments classify HOT1 as a positive regulator of telomere length. Furthermore, immunoprecipitation and cell fractionation analyses show that HOT1 associates with the active telomerase complex and promotes chromatin association of telomerase. Collectively, these findings suggest that HOT1 supports telomerase‐dependent telomere elongation.     

Unwinding protein complexes in ALTernative telomere maintenance

Telomeres are composed of specialized chromatin that includes DNA repair/recombination proteins, telomere DNA‐binding proteins and a number of three dimensional nucleic acid structures including G‐quartets and D‐loops. A number of studies suggest that the BLM and WRN recQ‐like helicases play important roles in recombination‐mediated mechanisms of telomere elongation or A lternative L engthening of T elomeres (ALT), processes that maintain/elongate telomeres in the absence of telomerase. BLM and WRN localize within ALT‐associated nuclear bodies in telomerase‐negative immortalized cell lines and interact with the telomere‐specific proteins POT1, TRF1 and TRF2. Helicase activity is modulated by these interactions. BLM functions in DNA double‐strand break repair processes such as non‐homologous end joining, homologous recombination‐mediated repair, resolution of stalled replication forks and synthesis‐dependent strand annealing, although its precise functions at the telomeres are speculative. WRN also functions in DNA replication, recombination and repair, and in addition to its helicase domain, includes an exonuclease domain not found in other recQ‐like helicases. The biochemical properties of BLM and WRN are, therefore, important in biological processes other than DNA replication, recombination and repair. In this review, we discuss some previous and recent findings of human rec‐Q‐like helicases and their role in telomere elongation during ALT processes. J. Cell. Biochem. 109: 7–15, 2010. © 2009 Wiley‐Liss, Inc.     

RPA prevents G‐rich structure formation at lagging‐strand telomeres to allow maintenance of chromosome ends

Replication protein A (RPA) is a highly conserved heterotrimeric single‐stranded DNA‐binding protein involved in DNA replication, recombination, and repair. In fission yeast, the Rpa1‐D223Y mutation provokes telomere shortening. Here, we show that this mutation impairs lagging‐strand telomere replication and leads to the accumulation of secondary structures and recruitment of the homologous recombination factor Rad52. The presence of these secondary DNA structures correlates with reduced association of shelterin subunits Pot1 and Ccq1 at telomeres. Strikingly, heterologous expression of the budding yeast Pif1 known to efficiently unwind G‐quadruplex rescues all the telomeric defects of the D223Y cells. Furthermore, in vitro data show that the identical D to Y mutation in human RPA specifically affects its ability to bind G‐quadruplex. We propose that RPA prevents the formation of G‐quadruplex structures at lagging‐strand telomeres to promote shelterin association and facilitate telomerase action at telomeres.     

Leishmania replication protein A-1 binds in vivo single-stranded telomeric DNA

Replication protein A (RPA) is a highly conserved heterotrimeric single-stranded DNA-binding protein involved in different events of DNA metabolism. In yeast, subunits 1 (RPA-1) and 2 (RPA-2) work also as telomerase recruiters and, in humans, the complex unfolds G-quartet structures formed by the 3' G-rich telomeric strand. In most eukaryotes, RPA-1 and RPA-2 bind DNA using multiple OB fold domains. In trypanosomatids, including Leishmania, RPA-1 has a canonical OB fold and a truncated RFA-1 structural domain. In Leishmania amazonensis, RPA-1 alone can form a complex in vitro with the telomeric G-rich strand. In this work, we show that LaRPA-1 is a nuclear protein that associates in vivo with Leishmania telomeres. We mapped the boundaries of the OB fold DNA-binding domain using deletion mutants. Since Leishmania and other trypanosomatids lack homologues of known telomere end binding proteins, our results raise questions about the function of RPA-1 in parasite telomeres.     

Differential arrival of leading and lagging strand DNA polymerases at fission yeast telomeres

To maintain genomic integrity, telomeres must undergo switches from a protected state to an accessible state that allows telomerase recruitment. To better understand how telomere accessibility is regulated in fission yeast, we analysed cell cycle‐dependent recruitment of telomere‐specific proteins (telomerase Trt1, Taz1, Rap1, Pot1 and Stn1), DNA replication proteins (DNA polymerases, MCM, RPA), checkpoint protein Rad26 and DNA repair protein Nbs1 to telomeres. Quantitative chromatin immunoprecipitation studies revealed that MCM, Nbs1 and Stn1 could be recruited to telomeres in the absence of telomere replication in S‐phase. In contrast, Trt1, Pot1, RPA and Rad26 failed to efficiently associate with telomeres unless telomeres are actively replicated. Unexpectedly, the leading strand DNA polymerase ε (Polε) arrived at telomeres earlier than the lagging strand DNA polymerases α (Polα) and δ (Polδ). Recruitment of RPA and Rad26 to telomeres matched arrival of DNA Polε, whereas S‐phase specific recruitment of Trt1, Pot1 and Stn1 matched arrival of DNA Polα. Thus, the conversion of telomere states involves an unanticipated intermediate step where lagging strand synthesis is delayed until telomerase is recruited.     

Regulation of the Rev1–pol ζ complex during bypass of a DNA interstrand cross‐link

DNA interstrand cross‐links (ICLs) are repaired in S phase by a complex, multistep mechanism involving translesion DNA polymerases. After replication forks collide with an ICL, the leading strand approaches to within one nucleotide of the ICL (“approach”), a nucleotide is inserted across from the unhooked lesion (“insertion”), and the leading strand is extended beyond the lesion (“extension”). How DNA polymerases bypass the ICL is incompletely understood. Here, we use repair of a site‐specific ICL in Xenopus egg extracts to study the mechanism of lesion bypass. Deep sequencing of ICL repair products showed that the approach and extension steps are largely error‐free. However, a short mutagenic tract is introduced in the vicinity of the lesion, with a maximum mutation frequency of ~1%. Our data further suggest that approach is performed by a replicative polymerase, while extension involves a complex of Rev1 and DNA polymerase ζ. Rev1–pol ζ recruitment requires the Fanconi anemia core complex but not FancI–FancD2. Our results begin to illuminate how lesion bypass is integrated with chromosomal DNA replication to limit ICL repair‐associated mutagenesis.     

The Pif1 family helicase Pfh1 facilitates telomere replication and has an RPA-dependent role during telomere lengthening

Pif1 family helicases are evolutionary conserved 5′–3′ DNA helicases. Pfh1, the sole Schizosaccharomyces pombe Pif1 family DNA helicase, is essential for maintenance of both nuclear and mitochondrial DNAs. Here we show that its nuclear functions include roles in telomere replication and telomerase action. Pfh1 promoted semi-conservative replication through telomeric DNA, as replication forks moved more slowly through telomeres when Pfh1 levels were reduced. Unlike other organisms, S. pombe cells overexpressing Pfh1 displayed markedly longer telomeres. Because this lengthening occurred in the absence of homologous recombination but not in a replication protein A mutant (rad11-D223Y) that has defects in telomerase function, it is probably telomerase-mediated. The effects of Pfh1 on telomere replication and telomere length are likely direct as Pfh1 exhibited high telomere binding in cells expressing endogenous levels of Pfh1. These findings argue that Pfh1 is a positive regulator of telomere length and telomere replication.     

Contributions of recombination and repair proteins to telomere maintenance in telomerase‐positive and negative Ustilago maydis

Homologous recombination and repair factors are known to promote both telomere replication and recombination‐based telomere extension. Herein, we address the diverse contributions of several recombination/repair proteins to telomere maintenance in Ustilago maydis, a fungus that bears strong resemblance to mammals with respect to telomere regulation and recombination mechanisms. In telomerase‐positive U. maydis, deletion of rad51 and blm separately caused shortened but stably maintained telomeres, whereas deletion of both engendered similar telomere loss, suggesting that the repair proteins help to resolve similar problems in telomere replication. In telomerase‐negative cells, the loss of Rad51 or Brh2 caused accelerated senescence and failure to generate survivors on semi‐solid medium. However, slow growing survivors can be isolated through continuous liquid culturing, and these survivors exhibit type II‐like as well as ALT‐like telomere features. In contrast, the trt1Δ blmΔ double mutant gives rise to survivors as readily as the trt1Δ single mutant, and like the single mutant survivors, exhibit almost exclusively type I‐like telomere features. In addition, we observed direct physical interactions between Blm and two telomere‐binding proteins, which may thus recruit or regulate Blm at telomeres. Our findings provide the basis for further analyzing the interplays between telomerase, telomere replication, and telomere recombination.     

Limited TTP supply affects telomere length regulation in a telomerase-independent fashion

An adequate supply of nucleotides is essential for DNA replication and DNA repair. Moreover, inhibition of TTP synthesis can cause cell death by a poorly characterized mechanism called thymine-less death. In the yeast Saccharomyces cerevisiae, the genes encoding thymidylate synthetase (CDC21) and thymidylate kinase (CDC8) are both essential for de novo TTP synthesis. The effects of temperature-sensitive mutations in these genes have been characterized and, curiously, the phenotypes displayed by cells harboring them include shortened telomeric repeat tracts. This finding raised the possibility that the enzyme telomerase is very sensitive to TTP-pools. We tested this possibility in vivo by assessing telomerase-dependent extension in situations of lowered TTP supply. The results show that the above-mentioned short telomere phenotype is not a consequence of an inability of telomerase to elongate telomeres when TTP synthesis is impaired. Moreover, this telomere shortening was abolished in cells harboring a mutation in DNA polymerase α. Previously, this same mutation was shown to affect the coordination between conventional replication and telomerase-mediated extension. These results thus re-emphasize the importance of the interplay between conventional replication and telomerase-mediated addition of telomeric repeats in telomere replication.     

Tel1ATM dictates the replication timing of short yeast telomeres

Telomerase action is temporally linked to DNA replication. Although yeast telomeres are normally late replicating, telomere shortening leads to early firing of subtelomeric DNA replication origins. We show that double‐strand breaks flanked by short telomeric arrays cause origin firing early in S phase at late‐replicating loci and that this effect on origin firing time is dependent on the Tel1^ATM checkpoint kinase. The effect of Tel1^ATM on telomere replication timing extends to endogenous telomeres and is stronger than that elicited by Rif1 loss. These results establish that Tel1^ATM specifies not only the extent but also the timing of telomerase recruitment.     

TPP1 as a versatile player at the ends of chromosomes

Telomeres, the ends of linear eukaryotic chromosomes, are tandem DNA repeats and capped by various telomeric proteins. These nucleoprotein complexes protect telomeres from DNA damage response （DDR）, recombination, and end-to-end fusions, ensuring genome stability. The human telosome/shelterin complex is one of the best-studied telomere-associated protein complexes, made up of six core telomeric proteins TRF1, TRF2, TIN2, RAPI, POT1, and TPPI. TPP1, also known as adrenocortical dysplasia protein homolog （ACD）, is a putative mammalian homolog of TEBP-β and belongs to the oligonucleotide binding （OB）-fold-containing protein family. Three functional domains have been identified within TPP1, the N-terminal OB fold, the POT1 binding recruitment domain （RD）, and the carboxyl-terminal TIN2-interacting domain （TID）. TPP1 can interact with both POT1 and TIN2 to maintain telomere structure, and mediate telomerase recruitment for telomere elongation. These features have indicated TPP1 play an essential role in telomere maintenance. Here, we will review important findings that highlight the functional significance of TPP1, with a focus on its interaction with other telosome components and the telomerase. We will also discuss potential implications in disease therapies.     

The mechanism of Single strand binding protein–RecG binding: Implications for SSB interactome function

The Escherichia coli single‐strand DNA binding protein (SSB) is essential to viability where it functions to regulate SSB interactome function. Here it binds to single‐stranded DNA and to target proteins that comprise the interactome. The region of SSB that links these two essential protein functions is the intrinsically disordered linker. Key to linker function is the presence of three, conserved PXXP motifs that mediate binding to oligosaccharide‐oligonucleotide binding folds (OB‐fold) present in SSB and its interactome partners. Not surprisingly, partner OB‐fold deletions eliminate SSB binding. Furthermore, single point mutations in either the PXXP motifs or, in the RecG OB‐fold, obliterate SSB binding. The data also demonstrate that, and in contrast to the view currently held in the field, the C‐terminal acidic tip of SSB is not required for interactome partner binding. Instead, we propose the tip has two roles. First, and consistent with the proposal of Dixon, to regulate the structure of the C‐terminal domain in a biologically active conformation that prevents linkers from binding to SSB OB‐folds until this interaction is required. Second, as a secondary binding domain. Finally, as OB‐folds are present in SSB and many of its partners, we present the SSB interactome as the first family of OB‐fold genome guardians identified in prokaryotes.     

Recruitment and positioning determine the specific role of the XPF‐ERCC1 endonuclease in interstrand crosslink repair

XPF‐ERCC1 is a structure‐specific endonuclease pivotal for several DNA repair pathways and, when mutated, can cause multiple diseases. Although the disease‐specific mutations are thought to affect different DNA repair pathways, the molecular basis for this is unknown. Here we examine the function of XPF‐ERCC1 in DNA interstrand crosslink (ICL) repair. We used Xenopus egg extracts to measure both ICL and nucleotide excision repair, and we identified mutations that are specifically defective in ICL repair. One of these separation‐of‐function mutations resides in the helicase‐like domain of XPF and disrupts binding to SLX4 and recruitment to the ICL. A small deletion in the same domain supports recruitment of XPF to the ICL, but inhibited the unhooking incisions most likely by disrupting a second, transient interaction with SLX4. Finally, mutation of residues in the nuclease domain did not affect localization of XPF‐ERCC1 to the ICL but did prevent incisions on the ICL substrate. Our data support a model in which the ICL repair‐specific function of XPF‐ERCC1 is dependent on recruitment, positioning and substrate recognition.     

The Pif1 Helicase,a Negative Regulator of Telomerase,Acts Preferentially at Long Telomeres

Telomerase, the enzyme that maintains telomeres, preferentially lengthens short telomeres. The S. cerevisiae Pif1 DNA helicase inhibits both telomerase-mediated telomere lengthening and de novo telomere addition at double strand breaks (DSB). Here, we report that the association of the telomerase subunits Est2 and Est1 at a DSB was increased in the absence of Pif1, as it is at telomeres, suggesting that Pif1 suppresses de novo telomere addition by removing telomerase from the break. To determine how the absence of Pif1 results in telomere lengthening, we used the single telomere extension assay (STEX), which monitors lengthening of individual telomeres in a single cell cycle. In the absence of Pif1, telomerase added significantly more telomeric DNA, an average of 72 nucleotides per telomere compared to the 45 nucleotides in wild type cells, and the fraction of telomeres lengthened increased almost four-fold. Using an inducible short telomere assay, Est2 and Est1 no longer bound preferentially to a short telomere in pif1 mutant cells while binding of Yku80, a telomere structural protein, was unaffected by the status of the PIF1 locus. Two experiments demonstrate that Pif1 binding is affected by telomere length: Pif1 (but not Yku80) -associated telomeres were 70 bps longer than bulk telomeres, and in the inducible short telomere assay, Pif1 bound better to wild type length telomeres than to short telomeres. Thus, preferential lengthening of short yeast telomeres is achieved in part by targeting the negative regulator Pif1 to long telomeres.     

G-quadruplex preferentially forms at the very 3' end of vertebrate telomeric DNA

Human chromosome ends are protected with kilobases repeats of TTAGGG. Telomere DNA shortens at replication. This shortening in most tumor cells is compensated by telomerase that adds telomere repeats to the 3′ end of the G-rich telomere strand. Four TTAGGG repeats can fold into G-quadruplex that is a poor substrate for telomerase. This property has been suggested to regulate telomerase activity in vivo and telomerase inhibition via G-quadruplex stabilization is considered a therapeutic strategy against cancer. Theoretically G-quadruplex can form anywhere along the long G-rich strand. Where G-quadruplex forms determines whether the 3′ telomere end is accessible to telomerase and may have implications in other functions telomere plays. We investigated G-quadruplex formation at different positions by DMS footprinting and exonuclease hydrolysis. We show that G-quadruplex preferentially forms at the very 3′ end than at internal positions. This property provides a molecular basis for telomerase inhibition by G-quadruplex formation. Moreover, it may also regulate those processes that depend on the structure of the very 3′ telomere end, for instance, the alternative lengthening of telomere mechanism, telomere T-loop formation, telomere end protection and the replication of bulky telomere DNA. Therefore, targeting telomere G-quadruplex may influence more telomere functions than simply inhibiting telomerase.     

Break-Induced Replication Requires DNA Damage-Induced Phosphorylation of Pif1 and Leads to Telomere Lengthening

Broken replication forks result in DNA breaks that are normally repaired via homologous recombination or break induced replication (BIR). Mild insufficiency in the replicative ligase Cdc9 in budding yeast Saccharomyces cerevisiae resulted in a population of cells with persistent DNA damage, most likely due to broken replication forks, constitutive activation of the DNA damage checkpoint and longer telomeres. This telomere lengthening required functional telomerase, the core DNA damage signaling cascade Mec1-Rad9-Rad53, and the components of the BIR repair pathway – Rad51, Rad52, Pol32, and Pif1. The Mec1-Rad53 induced phosphorylation of Pif1, previously found necessary for inhibition of telomerase at double strand breaks, was also important for the role of Pif1 in BIR and telomere elongation in cdc9-1 cells. Two other mutants with impaired DNA replication, cdc44-5 and rrm3Δ, were similar to cdc9-1: their long telomere phenotype was dependent on the Pif1 phosphorylation locus. We propose a model whereby the passage of BIR forks through telomeres promotes telomerase activity and leads to telomere lengthening.     

Dna2 Is Involved in CA Strand Resection and Nascent Lagging Strand Completion at Native Yeast Telomeres

Post-replicational telomere end processing involves both extension by telomerase and resection to produce 3′-GT-overhangs that extend beyond the complementary 5′-CA-rich strand. Resection must be carefully controlled to maintain telomere length. At short de novo telomeres generated artificially by HO endonuclease in the G₂ phase, we show that dna2-defective strains are impaired in both telomere elongation and sequential 5′-CA resection. At native telomeres in dna2 mutants, GT-overhangs do clearly elongate during late S phase but are shorter than in wild type, suggesting a role for Dna2 in 5′-CA resection but also indicating significant redundancy with other nucleases. Surprisingly, elimination of Mre11 nuclease or Exo1, which are complementary to Dna2 in resection of internal double strand breaks, does not lead to further shortening of GT-overhangs in dna2 mutants. A second step in end processing involves filling in of the CA-strand to maintain appropriate telomere length. We show that Dna2 is required for normal telomeric CA-strand fill-in. Yeast dna2 mutants, like mutants in DNA ligase 1 (cdc9), accumulate low molecular weight, nascent lagging strand DNA replication intermediates at telomeres. Based on this and other results, we propose that FEN1 is not sufficient and that either Dna2 or Exo1 is required to supplement FEN1 in maturing lagging strands at telomeres. Telomeres may be among the subset of genomic locations where Dna2 helicase/nuclease is essential for the two-nuclease pathway of primer processing on lagging strands.     

High-affinity RNA binding by a hyperthermophilic single-stranded DNA-binding protein

Single-stranded DNA-binding proteins (SSBs), including replication protein A (RPA) in eukaryotes, play a central role in DNA replication, recombination, and repair. SSBs utilise an oligonucleotide/oligosaccharide-binding (OB) fold domain to bind DNA, and typically oligomerise in solution to bring multiple OB fold domains together in the functional SSB. SSBs from hyperthermophilic crenarchaea, such as Sulfolobus solfataricus, have an unusual structure with a single OB fold coupled to a flexible C-terminal tail. The OB fold resembles those in RPA, whilst the tail is reminiscent of bacterial SSBs and mediates interaction with other proteins. One paradigm in the field is that SSBs bind specifically to ssDNA and much less strongly to RNA, ensuring that their functions are restricted to DNA metabolism. Here, we use a combination of biochemical and biophysical approaches to demonstrate that the binding properties of S. solfataricus SSB are essentially identical for ssDNA and ssRNA. These features may represent an adaptation to a hyperthermophilic lifestyle, where DNA and RNA damage is a more frequent event.

     

Structure of the Human Telomeric Stn1-Ten1 Capping Complex

The identification of the human homologue of the yeast CST in 2009 posed a new challenge in our understanding of the mechanism of telomere capping in higher eukaryotes. The high-resolution structure of the human Stn1-Ten1 (hStn1-Ten1) complex presented here reveals that hStn1 consists of an OB domain and tandem C-terminal wHTH motifs, while hTen1 consists of a single OB fold. Contacts between the OB domains facilitate formation of a complex that is strikingly similar to the replication protein A (RPA) and yeast Stn1-Ten1 (Ten1) complexes. The hStn1-Ten1 complex exhibits non-specific single-stranded DNA activity that is primarily dependent on hStn1. Cells expressing hStn1 mutants defective for dimerization with hTen1 display elongated telomeres and telomere defects associated with telomere uncapping, suggesting that the telomeric function of hCST is hTen1 dependent. Taken together the data presented here show that the structure of the hStn1-Ten1 subcomplex is conserved across species. Cell based assays indicate that hTen1 is critical for the telomeric function of hCST, both in telomere protection and downregulation of telomerase function.