And Mutl-Related Heterocomplexes

Commentary

Multiple functions of MutS- and MutL-related heterocomplexes

Takuro Nakagawa, Abhijit Datta, and Richard D. Kolodner*

Ludwig Institute for Cancer Research, Department of Medicine and Cancer Center, University of California San Diego School of Medicine, CMME 3080, 9500 Gilman Drive, La Jolla, CA 92093-0660

eterodimeric complexes of MutS- paired in msh4, zip1, and mer3 mutants (3, from zygotene (synapsis is started) Hand MutL-related proteins were 9, 14). zip1, zip2, and mer3 mutations also through pachytene (homologs are fully identified from studies of DNA mismatch cause cell-cycle arrest before meiosis I. synapsed) in oocytes (21–23). In contrast, repair (MMR) and have been implicated Although homolog nondisjunction is a pri- MLH1 is detected only at pachytene in in processing of recombination interme- mary cause of missegregation in crossing- spermatocytes. Although human PMS1 is diates. In a recent issue of PNAS, Wang et over-deficient mutants, precocious sepa- most closely related to the yeast MLH3 al. (1) have reported several observations ration of sister chromatids has also been and interacts with MLH1, it is also highly about the function of MutL-related genes observed in msh4, msh5, and zip1 mutants. related to yeast MLH2 (12, 24). Interest- of the yeast Saccharomyces cerevisiae in ZIP1 localizes continuously along the ingly, Pms1Ϫ͞Ϫ mice are fertile, suggesting MMR and recombination in meiosis. entire length of meiotic chromosomes few, if any, meiotic defects (25). Human First, they have observed that MLH1 is the when homologs are synapsed, which de- PMS2 (the yeast PMS1 homolog) forms a common subunit in three different MutL- pends on ZIP2 (7, 8). However, before heterodimer with MLH1, and Pms2Ϫ͞Ϫ related heterodimeric complexes, MLH1– synapsis, ZIP1 and ZIP2 are colocalized as mice show only male infertility (26, 27). PMS1, MLH1–MLH2, and MLH1– discrete foci on meiotic chromosomes. These findings raise questions about the MLH3. Second, a possible role of MLH1– ZIP2 also colocalizes with MRE11, a com- identity of the mammalian MLH3 ho- MLH2 in MMR was detected. Third, and ponent of the MRE11–RAD50–XRS2 molog and possibly suggest some redun- most striking, MLH1–MLH3 was shown complex, which is required for double- dancy between mammalian PMS1 and to play an important role in promoting strand break (DSB) formation and resec- PMS2. However, involvement of murine Ј crossing-over. This last observation ex- tion of the 5 ends (for a review, see ref. Msh5 and Mlh1 in chromosome synapsis in tends the eukaryotic paradigm of combi- 15). In addition, ZIP2 localization de- meiosis implies a conserved mechanism of natorial interactions between MutS- and pends on DSB formation. These results crossing-over. MutL-related heterodimeric complexes suggest that ZIP2 localizes at sites of and mispaired bases to other types of recombination. MSH4 has also been Combinatorial Specificity of MutS and MutL DNA structures formed during replica- shown to localize to similar discrete foci Homologs. Heterodimeric complexes of tion, recombination, and repair. on meiotic chromosomes (4). In zip1 and MutS- and MutL-related proteins were mer3 mutants, DSBs are not completely first observed in studies of eukaryotic Genetics of Crossover Regulation. In most converted to later intermediates, and pro- MMR (for reviews, see refs. 28–30). Ex- organisms, meiotic crossing-over of ho- gression of meiosis is delayed or blocked tensive studies have defined at least six mologous chromosomes (homologs) and (9, 16). Cell-cycle arrest occurs in zip2 different heterodimeric complexes that in- cohesion between sister chromatids are mutants, although the kinetics of DSB teract in different combinations (Fig. 1). required for faithful segregation of chro- repair has not been studied (8, 17). The Combinations of these different com- mosomes (2, 3). Several yeast genes, in- available data suggest that MSH4, MSH5, plexes apparently direct the processing of cluding MutS- and MutL-related genes, MLH1, MLH3, ZIP1, ZIP2, MER3, and different types of DNA structures. are required for normal levels of crossing- EXO1 all function at a similar time in Two MutS-related heterodimeric com- over but not for gene conversion; these meiosis and that defects in these genes plexes, MSH2–MSH6 and MSH2–MSH3, include MSH4, MSH5, MLH1, MLH3, cause similar defects in crossing-over. have been found to function in the repair ZIP1, ZIP2, MER3, and EXO1 (refs. 1 and MSH4, MSH5, and MLH1 homologs of mispaired bases generated during DNA 4–9; H. Tsubouchi and H. Ogawa, per- have been found in higher eukaryotes. replication and recombination. It seems sonal communication). Mutations in these Human MSH4 and MSH5 proteins form a that MSH2–MSH6 interacts with base͞ genes reduce crossing-over by 50% or heterodimeric complex (11, 13). Msh5Ϫ͞Ϫ Ϫ͞Ϫ base and insertion͞deletion mispairs, more, resulting in increased levels of ho- or Mlh1 mutant mice show male and whereas MSH2–MSH3 interacts only with molog nondisjunction and spore death. female infertility (18–21). Asynapsis insertion͞deletion mispairs (for a review, Genetic analyses have shown that MSH4, and͞or synapsis between nonhomologs see ref. 30). These complexes function in MSH5, and MLH1 are in the same epista- followed by apoptotic cell death before or conjunction with MutL-related het- sis group with regard to crossing-over, during pachytene are observed in these Ϫ͞Ϫ erodimeric complexes and with other in- indicating that crossing-over in these mu- mutant mice. In Mlh1 mice, high levels teracting proteins such as EXO1 and tants is not due to functional redundancy of univalents are seen as homologs desyn- PCNA (31–36). MLH1–PMS1, a MutL- among these genes (5, 6). The formation apse in the spermatocytes that do not of MSH4–MSH5 and MLH1–MLH3 het- undergo apoptosis, indicating that Mlh1 is erodimeric complexes has also been es- required for the formation or stabilization See companion article on page 13914 in issue 24 of tablished (1, 10–13). The regulation of the of chiasmata, the cytological manifesta- volume 96. distribution of crossovers along a chromo- tion of crossing-over. The MLH1 protein *To whom reprint requests should be addressed. E-mail: some, crossover interference, is also im- localizes to discrete foci on chromosomes [email protected].

14186–14188 ͉ PNAS ͉ December 7, 1999 ͉ vol. 96 ͉ no. 25 Downloaded by guest on September 30, 2021 and may involve direct recognition of Hol- liday junctions by MSH2–MSH6 (45–48). Interestingly, processing of SSA interme- diates involves MSH2–MSH3 and RAD1– RAD10, which interact with each other, but does not involve a MutL-related com- plex (49, 50). Regulation of crossing-over requires MSH4–MSH5 and MLH1–MLH3 (1, 4–6). However, it is not known whether these protein complexes interact with each other or whether MSH4–MSH5 interacts with DNA. Interactions with other pro- teins involved in regulating crossing-over have not been investigated, but logical candidates for such proteins include ZIP1, ZIP2, MER3, and EXO1. The above data raise the interesting possibility that the different MutS-related complexes reflect either divergent evolu- tion of protein complexes that recognize different DNA structures or a combina- torial mechanism for generating different DNA recognition specificity. Further Fig. 1. Combinatorial specificities of MutS- and MutL-related heterocomplexes. (a) Base͞base mispairs; specificity is generated through interac- ͞ Ј (b) insertion deletion mispairs; (c)5 tailed DNA structures generated by single-strand DNA annealing tions with other accessory factors such as (SSA) recombination; (d) Holliday junctions. Note that the DNA binding specificity for MSH4–MSH5 and its interaction with MLH1–MLH3 have not been shown. MLH1–MLH2 is not indicated, because it is not clear the MutL-related complexes and other with which MutS-related complex it interacts. proteins like RAD1–RAD10 and EXO1. The end result is a diverse set of mecha- nisms that direct the processing of DNA related complex, plays a major role in both vention of recombination between diver- structures generated during replication, the MSH2–MSH6 and MSH2–MSH3 gent DNA sequences, possibly by regulat- repair, and recombination. pathways and interacts with these two ing the extent of heteroduplex formation, Mechanistic Aspects of Meiotic Crossing- complexes (32, 36, 37). MLH1–MLH3, and the processing of SSA intermediates. Over. There are both early and late steps another MutL-related complex, also MSH2–MSH6, MSH2–MSH3, MLH1– during recombination at which proteins COMMENTARY seems to play a role in the repair of some PMS1, RAD1–RAD10, and EXO1 are like MSH4–MSH5 and MLH1–MLH3 mispaired bases recognized by MSH2– involved in preventing recombination be- might act (Fig. 2). That an early step might MSH3 (12). tween divergent DNA sequences (38–44). affect crossing-over can be visualized by MutS- and MutL-related proteins have The formation of gene conversion polarity examining the synthesis-dependent an- functions in recombination that are inde- gradients during meiotic recombination nealing model of DSB repair (for a review, pendent of MMR. These include the pre- requires MSH2–MSH6 and MLH1–PMS1 see ref. 51). In this model, the 3Ј end of one DNA strand invades the intact DNA partner and primes DNA synthesis. If this intermediate is unwound, single-strand annealing with the other half of the bro- ken chromosome can occur leading to gene conversion not associated with cross- ing-over. Alternatively, the invaded inter- mediate can be converted to intermedi- ates containing Holliday junctions, and crossovers or noncrossovers can result de- pending on how the junction is processed. Crossover control could occur early if the proteins required for crossing-over pro- mote the conversion of the single-end invasion intermediate to intermediates containing Holliday junctions. Alterna- tively, crossover control could occur at a late step if the protein required for cross- ing-over increases the Holliday junction cleavage in the orientation for the forma- tion of crossovers (4, 5). It is unclear whether MSH4–MSH5 and MLH1–MLH3 complexes act at Fig. 2. Steps of recombination and modes of the crossover control. (a) Resected DSBs; (b) strand invasion early or late steps in recombination. The of one DSB and 3Ј to 5Ј DNA synthesis (dashed line); (c) Unwound intermediates; (d)5Ј tailed DNA timing of recombination and levels of structures generated by SSA recombination; (e) Holliday junction intermediates. aberrant recombination intermediates in

Nakagawa et al. PNAS ͉ December 7, 1999 ͉ vol. 96 ͉ no. 25 ͉ 14187 Downloaded by guest on September 30, 2021 strains lacking these gene products are different distances between two junc- DSBs. msh4, msh5, mlh1, and mlh3 mu- not affected enough to cause pachytene tions) that are preferentially resolved as tants show reduced crossing-over but do arrest. However, mutations in other crossovers (52, 53). It is also possible that not show pachytene arrest. In contrast to Ϫ͞Ϫ Ϫ͞Ϫ genes encoding proteins that may func- there are two pathways: one that ac- yeast, in Mlh1 and Msh5 mice, tion along with MSH4–MSH5 and counts for 50% of crossovers and re- pachytene arrest and apoptosis occur. MLH1–MLH3, such as ZIP1 and MER3, quires the crossover control proteins dis- These occurrences could mean that re- cause defects in the transition of DSBs to cussed herein and another pathway that combination defects in the mutant mice later intermediates but otherwise cause is required for the rest of the crossovers are distinct from those in yeast or that the meiotic defects that are similar to those and noncrossovers. Clearly, additional checkpoint in mice is more sensitive. Al- caused by mutations in MSH4, MSH5, studies are required to answer this im- ternatively, this difference could reflect MLH1, and MLH3 (1, 4–7, 9, 16). Both portant question. different monitoring systems in the two Msh5Ϫ͞Ϫ and Mlh1Ϫ͞Ϫ mutations in mice species. For instance, recent observations decrease crossing-over and cause defects Links Between Meiotic Recombination and in mammalian cells have shown that in the progression of meiosis (18–23). It Checkpoint Control. Yeast mutants with de- MMR proteins may also serve as DNA is possible that the mouse mutations fective meiotic recombination and͞or damage sensors that trigger an apoptotic affect the same step(s) of recombination chromosome synapsis often execute a cell- response (57). The possible role of pro- affected in the corresponding yeast mu- cycle arrest. The mitotic checkpoint genes teins like MSH4–MSH5 and MLH1– tants. Overall, these findings are consis- RAD17, RAD24, MEC1, MEC3, and MLH3 as direct sensors of recombination tent with the idea that MSH4–MSH5 and DDC1 and the meiosis-specific genes intermediates will be an interesting area MLH1–MLH3 promote DSB processing RED1, MEK1, PCH2, and MSC6 are in- for future investigation. to form Holliday junctions reducing al- volved in this cell-cycle arrest (17, 54–56). ternative outcomes, such as SSA recom- The signals that initiate meiotic check- We wish to thank Hideo Tsubouchi, Hideyuki bination, rather than to alter the resolu- points are unclear but may include DSBs Ogawa, Sue Jinks-Robertson, Gray F. Crouse, tion of Holliday junctions. Such an early and other recombination intermediates as and Josef Jiricny for sharing data before pub- lication and Douglas K. Bishop and Alex R. function could include promoting the well as chromosome synapsis. zip1, zip2, Shoemaker for suggestions and comments on formation of specific-types of Holliday and mer3 yeast mutants show reduced the manuscript. Related work by the authors junctions (e.g., geometrically distinct crossing-over and pachytene arrest, possi- has been supported by National Institutes of types of double Holliday junctions or bly because of delayed processing of Health Grants GM26017 and GM50006.

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