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Holliday Junctions and Resolution

Most models for crossover formation include one or more Holliday junctions - four-stranded structures involving a partner swap between two duplex DNA molecules. In these models, crossovers are actually made by 'resolving' the HJs through cleavage of two strands with the same polarity; enzymes that do this are called resolvases. Three nuclear enzymes have been shown to cleave HJs in vitro: Mus81-Mms4/Eme1, GEN1/Yen1, and SLX4-SLX1. These are described below, along with what wse believe is a fourth resolvse - mei-HEM.
 

S. pombe Mus81-Eme1 was the first candidate nuclear resolvase (Boddy 2001). This enzyme actually cuts nicked Holliday junctions and replication fork structures better than HJs (Gaillard 2003). When it cuts HJs, the ends cannot be ligated back together like canonical resolvases - hence, our choice of a chainsaw to represent this enzyme. This has led some to suggest that Mus81-Eme1 is not a true resolvase (e.g., Ehmsen 2008); however, genetic evidence, especially from S. pombe, suggests that HJs might be the in vivo target in generating meiotic crossovers (Cromie 2006).

Drosophila MUS81-MMS4 does not seem to have any function in generating meiotic crossovers. Flies don't seem to care whether they have this enzyme or not (mus81 mutants even seem to grow better than wild-type flies), unless the DmBLM helicase is also absent - then they're inviable.

Human GEN1 and S. cerevisiae Yen1 are true resolvases in vitro (Ip 2008), cut HJs with high specificity and with precision (as can be predicted from the structure shown here). In vivo, however, they seem to play second fiddle to Mus81-Mms4/Eme1 (Blanco 2010, Tay 2010, Ho 2010). S. pombe doesn't even have a GEN1/Yen1 ortholog.
 
Drosophila seems to have reversed this situation: GEN is more important than MUS81-MMS4. Gen mutants have severe hypersensitivities to DNA damaging agents (unlike mus81 mutants, which are only moderately sensitive to a limited number of agents) and mutants that lack both GEN and the DmBLM helicase dies very early in development (mutants lacking MUS81 and DmBLM die very late). We are continuing to explore functions of GEN in vivo and in vitro.

S.L. Andersen, H.K. Kuo, D. Savukoski, M.H. Brodsky, and J. Sekelsky (2011) Three structure-selective endonucleases are essential in the absence of BLM helicase in Drosophila. PLoS Genetics 7: e1002315.

SLX4-SLX1 is the most recently publisahed resolvase (Fekairi 2009, Muñoz 2009, Svendsen 2009). This enzyme seems to be involved in repairing DNA inter-strand crosslinks, a function that is underscored by the finding that mutations in SLX4 cause Fanconi anemia (Kim 2011, Stoepker 2011).

SLX4/MUS312 is the Swiss army knife of nuclease scaffolding proteins. We first discovered Drosophila MUS312 as a protein that interacts with the nuclease MEI-9. This interaction is essential for generating meiotic crossovers. At the time, MUS312 was a pioneer protein; we now know it is orthologous to SLX4/BTBD12, although there is very little similarity at the amino acid level. As with the vertebrate proteins, MUS312-SLX1 appears to be important in inter-strand crosslink repair. As with the fungal proteins, MUS312-SLX1 is essential when the BLM helicase is absent. Studies of this lethality has suggested functions for both the resolvase and the helicase in replication fork repair. We are now testing these hypotheses.

Ö. Yildiz, S. Majumder, B.C. Kramer, and J. Sekelsky (2002) Drosophila MUS312 protein interacts physically with the nucleotide excision repair endonuclease MEI-9 to generate meiotic crossovers. Molecular Cell 10: 1503-1509.

S.L. Andersen, D.T. Bergstralh, K.P. Kohl, J.R. LaRocque, C.B. Moore, and J. Sekelsky (2009) Drosophila MUS312 and the vertebrate ortholog BTBD12 interact with DNA structure-specific endonucleases in DNA repair and recombination. Molecular Cell 35: 128-135.

S.L. Andersen, H.K. Kuo, D. Savukoski, M.H. Brodsky, and J. Sekelsky (2011) Three structure-selective endonucleases are essential in the absence of BLM helicase in Drosophila. PLoS Genetics 7: e1002315.
 

mei-9 and Ercc1 encode the Drosophila orthologs of S. cerevisiae Rad1p and Rad10p, and mammalian XPF/ERCC4 and ERCC1. The heterodimer is an endonuclease that recognizes and cleaves DNA strands that transit from 5' double-stranded to 3' single-stranded, such as bubbles generated during nucleotide excision repair (NER) (Bardwell 1994, Park 1995). mei-9 and Ercc1 mutants are hypersensitive to many types of DNA damaging agents, including ionizing radiation, ultraviolet radiation, and interstrand crosslinking agents. These hypersensitivities underlie some of the pathways in which the endonuclease functions, including NER and ICL repair. Interestingly, mei-9 and Ercc1 mutants also have defects in meiotic recombination, including a 90% decrease in crossovers. We hypothesized that MEI-9-ERCC1 cuts Holliday junctions to generate crossovers, as shown in the animation at the top of this page.

In our model, in the absence of MEI-9 the double-HJ undergoes dissolution, as shown at right. Because there are no nicks made during dissolution, mismatch repair does not occur, leading to post-meiotic segregation (PMS) of markers, seen as mosaic progeny. Carpenter (1982) observed PMS in mei-9 mutants. We mapped PMS tracts and showed that they exhibit the "trans" arrangement predicted by the model.

S.J. Radford, S. McMahan, H. Blanton, and J. Sekelsky (2007) Heteroduplex DNA in meiotic recombination in Drosophila mei-9 mutants Genetics 176: 63-72.

Genetic studies identified two additional proteins implicated in generating crossovers with MEI-9-ERCC1: the saffolding protein MUS312 and the OB-fold protein HDM (Joyce 2009). We call the complex mei-HEM (MEI-9, HDM, ERCC1, MUS312). We've expressed this complex in insect cells using a baculovirus system and purified it, and are now conducting studies of its nuclease activities.

Ö. Yildiz, S. Majumder, B.C. Kramer, and J. Sekelsky (2002) Drosophila MUS312 protein interacts physically with the nucleotide excision repair endonuclease MEI-9 to generate meiotic crossovers. Molecular Cell 10: 1503-1509.