In this study, we show that ScMre11p displays substantially higher binding affinity for G4 DNA, compared with single- or double-stranded DNA. Importantly, Mre11p cleaves G-rich single-stranded and G4 DNA in a sequence- and structure-specific manner. Our results are consistent with the genetic evidence implicating S.cerevisiae MRE11
in the establishment of proper telomere end structure (29
). These observations suggest that ScMre11p might provide appropriate DNA termini for telomerase-mediated replication of telomeric DNA and loading of telomere-binding proteins.
Although elegant genetic and molecular biology approaches have led to the identification of components involved in telomere length maintenance, yet our understanding of how these proteins regulate telomere length is not clear. Several lines of evidence invoke MRX complex as the functional entity in regulation of telomere length. To begin to understand the mechanistic aspects of MRX function, we investigated the biochemical activities of Mre11p using DNA-binding and nuclease assays. We found that Mre11p displays higher binding affinity for G4 DNA, a configuration of DNA that is presumed to exist at telomeres (25
), compared with single- or double-stranded DNA. Further analysis demonstrated that Mre11p binds G4 DNA with 12-fold higher binding affinity over ssDNA. Surprisingly, Mre11p remains stably bound even in the presence of non-physiological concentrations of NaCl. The physiological significance of its stable binding to G4 DNA remains to be elucidated. The observation that Mre11p binds G4 DNA with higher binding affinity led us to a second line of inquiry concerning its intrinsic nuclease activity. To address this question, we examined the ability of Mre11p to cleave G4 DNA in the presence of Mn2+
. The endonucleolytic products generated by Mre11p with both single-stranded and G4 DNA contain G residue at the 5′ end. Thus, the ends generated by Mre11p could either directly serve as a template for telomerase or require further processing by other telomere-binding proteins.
, both telomerase-dependent and telomerase-independent mechanisms co-exist to ensure proper telomere length. Inactivation of the components of telomerase triggers progressive telomere shortening culminating in genomic instability and cell death (41
). In addition, telomerase-proficient cells but lacking any one or all three members of the MRX complex are non-viable and show severe defects in recombination and telomere length maintenance (6
). When the telomeres reach a critical short length, a recombination process mediated by Rad52p, MRX complex and other components leads to telomere elongation, thus enabling few cells to regain wild-type growth potential (6
). These survivors fall into two classes: type I cells that show amplification of the telomere-associated Y′ elements and have very short TG1–3
repeat tracts and type II cells that have long variable tracts of TG1–3
repeats and only modest Y′ element amplification (44
). Type I-mediated recombination requires RAD51
and the replicative polymerases, whereas type II survivors depend on RAD50
In Trypanosoma brucei
, mouse and human cells, telomeric DNA adopts T-loop structures (48
). Similarly, Taz1p of fission yeast catalyzes the formation of higher order structures and T-loops from model DNA substrates containing 3′ single-stranded telomeric overhangs (50
). Although a similar structure has not been reported for S.cerevisiae
, it is proposed that the formation of G-quartet and fold back structures may account for the repression of telomere proximal genes and stabilization of telomeres. S.cerevisiae
strains lacking telomerase lose their telomeres gradually and eventually perish due to chromosome instability (23
). It has been speculated that helicases disrupt G•G (Hoogsteen) base pairs in G4 DNA to generate 3′ TTAGGG overhangs, but the molecular mechanism of the generation of free-ends required for helicase(s) action is obscure (32
). Genetic studies have shown that MRX complex is required for the generation of G-strand and loading of G-strand binding proteins (51
). The most striking observation in this report is that Mre11p cleaves G4 DNA within the vicinity of G-quartets. In view of the fact that helicases and telomerase require free ends to serve substrates, these findings indicate that the endonucleolytic activity of Mre11p is likely to play a direct biological role in vivo
A role for ScMre11p in the cleavage of G4 DNA can be visualized in the context of replication of telomerase-dependent telomeric DNA. The generation of suitable substrate for telomerase and telomere-binding proteins present a unique challenge to cells. Short direct DNA repeats comprise the underlying telomeric DNA and the strand that runs 5′ to 3′ towards the chromosome end is rich in guanines (G-tail). G-tails are presumed substrates for telomerase. Studies in S.cerevisiae
have shown that telomeres gain long G-tails late in S phase after replication (50
). The steps involved in elongation of telomeres include the generation of single-stranded TG1–3
tails, Cdc13p binding to this tail, recruitment of telomerase, Est2p-mediated lengthening of the TG1–3
tail, and C-strand synthesis (23
). It has been speculated that Tel1p and MRX complex could act at any of these steps.
Careful inspection of the sites of telomerase addition revealed that healing at spontaneous breaks often takes place at naturally occurring G-rich seed sequences that resemble the G-rich repeats found at telomeres. This suggests that the sequence-specific telomere end-binding protein Cdc13, which is responsible for recruiting telomerase to chromosome ends (50
), may similarly mediate the access of telomerase to DSBs. In strains of certain genetic backgrounds, the spectrum of chromosome healing events and telomere addition display a strong bias for sites that contain G-rich tracts, with essentially no healing at sites that lack a G-rich seed (50
). Our data are consistent with a direct role for G-rich DNA endonuclease activity of ScMre11p in several nuclear processes including telomere length maintenance, cellular response to DNA damage and recombination.
Together, these results suggest that the nuclease activities of ScMre11p on G4 DNA and G-rich ssDNA are likely to be important for telomere replication and recombinational repair. Since the MRX complex is conserved, we speculate that a similar mechanistic role is likely to ensue for ScMre11p in vertebrate cells.