Tethering Recombination Initiation Proteins inSaccharomyces cerevisiaePromotes Double Strand Break Formation

Abstract

Meiotic recombination in Saccharomyces cerevisiae is initiated by the creation of DNA double strand breaks (DSBs), an event requiring 10 recombination initiation proteins. Published data indicate that these 10 proteins form three main interaction subgroups [(Spo11-Rec102-Rec104-Ski8), (Rec114-Rec107-Mei4), and (Mre11-Rad50-Xrs2)], but certain components from each subgroup may also interact. Although several of the protein-protein interactions have been defined, the mechanism for DSB formation has been challenging to define. Using a variation of the approach pioneered by others, we have tethered 8 of the 10 initiation proteins to a recombination coldspot and discovered that in addition to Spo11, 6 others (Rec102, Rec104, Ski8, Rec114, Rec107, and Mei4) promote DSB formation at the coldspot, albeit with different frequencies. Of the 8 proteins tested, only Mre11 was unable to cause DSBs even though it binds to UAS GAL at GAL2. Our results suggest there may be several ways that the recombination initiation proteins can associate to form a functional initiation complex that can create DSBs.

Analysis of meiotic recombination initiation in Saccharomyces cerevisiae

“…Several of the initiation proteins (i.e., Mre11, Spo11, Mei4, and Rec114) have been shown to be located specifically at the site of DSBs [i.e., hotspots; (Borde, Lin et al 2004;Prieler, Penkner et al 2005;Sasanuma, Murakami et al 2007)]. All ten are required for recombination initiation (Peciña, Smith et al 2002), and at least seven are able to target DSBs at a coldspot (Koehn, Haring et al 2009). These observations, in addition to the many experiments that suggest subcomplexes exist among the initiation proteins (see Introduction), support the idea that a bona fide recombination initiation complex exists.…”

“…The biochemical roles of Mre11 and Rad50 after DSB formation are relatively clear (Borde 2007), but their role in actually creating the DSB is still unknown. Mre11 associates with hotspots when all the other RIPs are present (Borde, Lin et al 2004), but from the tethering experiments presented in Chapter 2, it does not appear to be responsible for recruiting the rest of the RIPs to the DNA for initiation (Koehn, Haring et al 2009). We have not tested the ability of the other members of the MRX subcomplex (i.e.…”

“…Interactions have also been described based on dependency relationships of one protein on another for its association with DNA (or chromatin in general), or specifically with hotspot DNA. It is currently accepted that three main subcomplexes exist among the ten RIPs (the Spo11-subcomplex, the Rec114subcomplex, and the MRX-subcomplex), with interactions also occurring across subcomplexes Koehn, Haring et al 2009;Li, Hooker et al 2006;Maleki, Neale et al 2007;.…”

The signal between the initiation of recombination and the first division of meiosis in Saccharomyces cervisiae

“…Though the above evidence suggests an order of assembly of the recombination initiation complex, Demelza Koehn's work suggests that if this order of assembly occurs, then it is not absolutely essential for function (Koehn et al, 2009). She has shown that strains with DB fusions of SPO11, REC104, REC114, REC102, REC107, MEI4 and SKI8 can all make DSBs at GAL2, suggesting that each of these proteins are capable of recruiting and forming a functional recombination initiation complex, despite presumably altering the wild type order of assembly.…”

“…She found that DB-SKI8 strains made DSBs at GAL2, although not as many as DB-Spo11 (7.3% in DB-Spo11 vs. 2.2% in DB-Ski8). This indicates that SKI8 could recruit the other factors including SPO11 to the DNA and form a functional complex, suggesting that Ski8 may serve a critical role in complex formation other than being merely a scaffold protein that allows Spo11-Rec102-Rec104 association (Koehn et al, 2009).…”