CELl cycle Feature
CELl cycle Feature
Cell Cycle 10:16, 2607-2609; August 15, 2011; © 2011 Landes Bioscience
Double duty for Exo1 during meiotic recombination Neil Hunter Howard Hughes Medical Institute; Departments of Microbiology, Molecular and Cellular Biology and Cell Biology and Human Anatomy; University of California, Davis; Davis, CA USA
Key words: Exo1, nuclease, homologous recombination, meiosis, crossing over, double-strand break, Holliday junction, joint molecule, strand exchange Exonuclease 1 is an XPG/Rad2-family metallonuclease that functions in a variety of DNA maintenance pathways. Exo1 functions are best defined in DNA mismatch repair, where it excises the nascent mismatch-containing strand, and during double-strand break (DSB) repair, in which Exo1 resects DNA ends in preparation for homologous recombination. Recent studies have focused on the role of Exo1 in meiotic recombination, where it functions to promote crossing over. These studies reveal temporally and biochemically distinct functions for Exo1. First, Exo1 plays a non-redundant role in creating extensively resected DSBs. Exo1 also acts at the ultimate step of meiotic recombination to promote the resolution of double Holliday junctions into crossovers. This latter function does not require the nuclease activity of Exo1, but involves interaction with another mismatch repair component, MutLγ (Mlh1-Mlh3), which is a putative endonuclease. These observations support the idea that mismatch repair components function directly in the nucleolytic resolution of double Holliday junctions into crossovers. Exonuclease 1 (Exo1) is a member of the XPG/Rad2 5' metallonuclease family and processes a variety of DNA structures during DNA repair. Exo1 is best known for its role in DNA mismatch repair, but it is now clear that Exo1 functions in most aspects of DNA metabolism, including replication, homologous recombination, telomere maintenance and checkpoint signaling.1,2
Exo1 has two apparently distinct nuclease activities, acting as a 5'-3' exonuclease on DNA nicks, gaps and ends and acting as an endonuclease on 5'-flaps. However, a recent structural study unifies these two activities by a common enzymatic mechanism.2 Exo1 specifically binds its substrates by inducting a sharp (90°) bend at the 5' junction of the nick, gap or flap. For non-flap substrates, the terminal two bases at these kinked intermediates are then displaced or “frayed,” thereby mimicking tiny 5' flaps. Thus, all substrates are ultimately cleaved via a common, metaldependent hydrolytic center. The functions of Exo1 are now particularly well characterized in two different contexts: DNA mismatch repair and the processing of double-strand breaks during homologous recombination. During mismatch repair, Exo1 loads at a nick located 5' to the mismatched bases. Exo1 nuclease activity remains relatively inactive, possibly via autoinhibition, until stimulated by MutS proteins. A MutS heterodimer (MutSα = MSH2-MSH6 or MutSβ = MSH2-MSH3) specifically binds the DNA mismatch and subsequently converts into a sliding clamp that can diffuse along the DNA duplex. When the MutS clamp encounters Exo1, 5'-3' exonucleolytic activity is stimulated, and the nascent mismatch-containing strand is excised.2 The repair of DNA double-strand breaks (DSBs) by homologous recombination requires the formation of long 3'-tailed DSB ends, which act as substrates for assembly of nucleoprotein filaments
comprising the homologous pairing and DNA strand exchange protein, Rad51 (and its meiosis-specific paralog, Dmc1).3 Exo1 defines one of two apparently redundant pathways that can catalyze extensive 5'-3' DSB resection.4 In this context, the binding and processivity of Exo1 is stimulated by an ensemble of the Rad50-Mre11Xrs2 complex (Mre11-Rad50-Nbs1 in humans) plus Sae2 (human CtIP). Two recent studies focused on the essential role of Exo1 for meiotic crossing over.6,7 Exo1 falls into a class of factors that are required for high levels of crossing over but that are not essential for efficient DSB repair, per se.3 How Exo1 functions in this capacity has remained a mystery. The suspected role of Exo1 in meiotic DSB resection suggested a model in which the crossover or non-crossover outcome of meiotic recombination is regulated by the length of the single-strand tails.5 Crossover and non-crossover pathways bifurcate after initial strand exchange forms nascent joint molecule intermediates called displacement loops (D-loops) (Fig. 1). Along the crossover pathway, metastable intermediates called Single-End Invasions (SEIs) and double Holliday Junctions (dHJs) are formed. dHJs are then resolved into crossovers via an unknown mechanism. Non-crossovers are thought to arise primarily via the synthesis-dependent strandannealing (SDSA) mechanism, in which the D-loop primes DNA synthesis, and then the nascent strand is dissociated and annealed to the second end of the break (Fig. 1). Thus, the longer 3'-tails resulting
©201 1L andesBi os c i enc e. Donotdi s t r i but e.
Correspondence to: Neil Hunter; Email:
[email protected] Submitted: 05/02/11; Accepted: 05/12/11 DOI: 10.4161/cc.10.16.16452 Comment on: Zakharyevich K, et al. Mol Cell 2010; 40:1001–15. www.landesbioscience.com
Cell Cycle
2607
©201 1L andesBi os c i enc e. Donotdi s t r i but e. Figure 1. Temporally and biochemically distinct activities of Exo1 promote double-strand break resection and the resolution of double Holliday junctions into crossovers. Model of meiotic recombination showing the molecular steps at which Exo1 functions. DSB formation is catalyzed by the Spo11 transesterase. DSB resection is initiated via the endonuclease activity of the Mre11-Rad50-Xrs2-Sae2 ensemble, which also liberates Spo11-oligo complexes. The molecular phenotypes of exo1 mutants are indicated. Despite limited DSB resection, exo1Δ null mutants and nuclease-inactive exo1D173A mutants remain proficient for the formation of strand exchange intermediates. exo1Δ-null mutants, but not exo1-D173A cells, are defective for converting double Holliday junctions into crossovers.
from the action of Exo1 at DSB ends were expected to facilitate formation of more extensive and more stable strand exchange intermediates and thereby promote progression along the crossover pathway. In contrast, short DSB tails would result in more labile intermediates that are likely to result in non-crossover SDSA. Using two different techniques, Keelagher et al. and Zakharyevich et al. demonstrated a major non-redundant role for Exo1 in meiotic DSB resection.6-8 However, contrary to expectations, the limited DSB resection in exo1Δ mutants did not impede the formation of crossoverspecific dHJs (Fig. 1). This result points to a second, later role for Exo1 in meiotic recombination: promoting the resolution of dHJs into crossovers. This result was
2608
especially intriguing given that the identity of the nuclease(s) that resolves meiotic dHJs into crossovers remains unclear. However, analysis of nuclease-inactive point mutants indicated that the nucleolytic activity of Exo1 is dispensable for its meiotic crossover function revealing an unanticipated and biochemically distinct role for Exo1 in dHJ resolution. Previous studies have also hinted at nuclease-independent or “structural” functions of Exo1,1,9 although none as absolute as that seen for meiotic crossing over. For example, Amin et al. showed that the presence of Exo1 suppresses the mutator phenotypes of a number of non-null mutations located in the mismatch repair factors, Mlh1 and Msh2 (with which Exo1 interacts) as well as their binding
Cell Cycle
partners (which do not interact directly with Exo1).9 The behavior of these “exo1dependent mutator mutations” led the authors to propose that interaction with Exo1 helps maintain Mlh1 and Msh2 in a conformation that facilitates their interactions with other protein partners. Exactly how Exo1 promotes the resolution of dHJs into crossovers remains unknown and will require unambiguous identification of the nuclease(s) that resolves double Holliday junctions. However, the data of Zackharyevich et al. hint at a possible role for another mismatch repair factor, the MutLγ complex, Mlh1Mlh3.7 MutLγ has a well-established role in crossing over and specifically localizes to future crossover sites. Moreover, MutLγ is a putative endonculease and, as such, may
Volume 10 Issue 16
participate directly in dHJ resolution.10 Zackharyevich et al. demonstrated that the interaction between Exo1 and MutLγ is important for crossing over and suggested that Exo1 may stimulate the MutLγ endonuclease to incise Holliday junctions. Further extending the mismatch repair paradigm, crossing over also requires meiosis-specific MutS homologs, Msh4 and Msh5, which bind as a complex to recombination intermediates, including Holliday junctions.11 In vitro and in vivo data are consistent with Msh4-Msh5
sliding clamps embracing the recombining duplexes to stabilize strand exchange intermediates.11,12 Thus, Msh4-Msh5 appears to function at an intermediate step of meiotic recombination to facilitate the formation of crossover-specific intermediates. However, by analogy to mismatch repair, a role for Msh4-Msh5 at the time of dHJ resolution also seems likely. Verifying a role for the Msh4-Msh5-Exo1Mlh1-Mlh3 ensemble in meiotic Holliday junction resolution remains an important outstanding goal for the field.
References 1. 2. 3.
Tran PT, et al. DNA Repair 2004; 3:1549-59. Orans J, et al. Cell 2011; 145:212-23. Hunter N. In: Molecular Genetics of Recombination. Heidelberg: Springer-Verlag 2006; 381-442. 4. Mimitou EP, et al. DNA Repair 2011; 10:344-8. 5. Tsubouchi H, et al. Mol Biol Cell 2000; 11:2221-33. 6. Keelagher RE, et al. DNA Repair 2010; 10:126-37. 7. Zakharyevich K, et al. Mol Cell 2010; 40:1001-15. 8. Hodgson A, et al. DNA Repair 2010; 10:138-48. 9. Amin NS, et al. Mol Cell Biol 2001; 21:5142-55. 10. Nishant KT, et al. Genetics 2008; 179:747-55. 11. Snowden T, et al. Mol Cell 2004; 15:437-51. 12. Borner GV, et al. Cell 2004; 117:29-45.
©201 1L andesBi os c i enc e. Donotdi s t r i but e.
www.landesbioscience.com
Cell Cycle
2609
