Distinct functions of S. pombe Rec12 (Spo11) protein and Rec12-dependent crossover recombination (chiasmata) in meiosis I; and a requirement for Rec12 in meiosis II

BACKGROUND: In most organisms proper reductional chromosome segregation during meiosis I is strongly correlated with the presence of crossover recombination structures (chiasmata); recombination deficient mutants lack crossovers and suffer meiosis I nondisjunction. We report that these functions are separable in the fission yeast Schizosaccharomyces pombe. RESULTS: Intron mapping and expression studies confirmed that Rec12 is a member of the Spo11/Top6A topoisomerase family required for the formation of meiotic dsDNA breaks and recombination. rec12-117, rec12-D15 (null), and rec12-Y98F (active site) mutants lacked most crossover recombination and chromosomes segregated abnormally to generate aneuploid meiotic products. Since S. pombe contains only three chromosome pairs, many of those aneuploid products were viable. The types of aberrant chromosome segregation were inferred from the inheritance patterns of centromere linked markers in diploid meiotic products. The rec12-117 and rec12-D15 mutants manifest segregation errors during both meiosis I and meiosis II. Remarkably, the rec12-Y98F (active site) mutant exhibited essentially normal meiosis I segregation patterns, but still exhibited meiosis II segregation errors. CONCLUSIONS: Rec12 is a 345 amino acid protein required for most crossover recombination and for chiasmatic segregation of chromosomes during meiosis I. Rec12 also participates in a backup distributive (achiasmatic) system of chromosome segregation during meiosis I. In addition, catalytically-active Rec12 mediates some signal that is required for faithful equational segregation of chromosomes during meiosis II

The Basic Cleft of RPA70N Binds Multiple Checkpoint Proteins, Including RAD9, To Regulate ATR Signaling▿

ATR kinase activation requires the recruitment of the ATR-ATRIP and RAD9-HUS1-RAD1 (9-1-1) checkpoint complexes to sites of DNA damage or replication stress. Replication protein A (RPA) bound to single-stranded DNA is at least part of the molecular recognition element that recruits these checkpoint complexes. We have found that the basic cleft of the RPA70 N-terminal oligonucleotide-oligosaccharide fold (OB-fold) domain is a key determinant of checkpoint activation. This protein-protein interaction surface is able to bind several checkpoint proteins, including ATRIP, RAD9, and MRE11. RAD9 binding to RPA is mediated by an acidic peptide within the C-terminal RAD9 tail that has sequence similarity to the primary RPA-binding surface in the checkpoint recruitment domain (CRD) of ATRIP. Mutation of the RAD9 CRD impairs its localization to sites of DNA damage or replication stress without perturbing its ability to form the 9-1-1 complex or bind the ATR activator TopBP1. Disruption of the RAD9-RPA interaction also impairs ATR signaling to CHK1 and causes hypersensitivity to both DNA damage and replication stress. Thus, the basic cleft of the RPA70 N-terminal OB-fold domain binds multiple checkpoint proteins, including RAD9, to promote ATR signaling

SMARCAL1 and WRN co-localize at stalled replication forks.

<p>(A) HeLa cells expressing GFP-WRN were treated with 2 mM HU, fixed, and stained with antibodies to SMARCAL1. (B) HeLa cells were transfected with non-targeting (NT) or WRN siRNAs then treated with 2 mM HU for the indicated times. Cells were fixed and stained with antibodies to SMARCAL1. (C) GFP-WRN expressing HeLa cells were transfected with NT or SMARCAL1 siRNAs, treated with 2 mM HU for the indicated times, fixed, and imaged for WRN. No significant difference is observed between the NT and SMARCAL1 siRNA samples.</p

Identification of SMARCAL1 interacting proteins by mass spectrometry.

<p>(A) 293T cells were transfected with a Flag-SMARCAL1 expression vector or an empty vector control. Cells were harvested, lysed and Flag antibody conjugated beads were used to immunopurify SMARCAL1. HeLa cells expressing endogenous SMARCAL1 were harvested, lysed, and SMARCAL1-909 antibody or IgG control antibody were used for immunopurification. Proteins were eluted from beads with either the Flag or SMARCAL1-909 peptides and subjected to 2D-LC-tandem mass spectrometry. Where indicated, the cells were treated with 2 mM HU for 16 h prior to harvesting. The number of peptides identified in each purification is reported. (B–D) HeLa nuclear extracts were prepared. Control IgG, SMARCAL1-909 antibody, or WRN antibody immunoprecipitates (IP) as indicated were separated by SDS-PAGE and immunoblotted with the indicated antibodies. Where indicated the nuclear extracts were treated with benzonase prior to the immunoprecipitation. In (D) the cells were either mock treated (Unt.) or incubated with 2 mM HU for 16 h prior to harvesting.</p

RPA acts as a scaffold to mediate the SMARCAL1-WRN interaction.

<p>(A) GST-WRN proteins bound to glutathione beads were incubated with HeLa nuclear extracts. After extensive washing, bound proteins were separated by SDS-PAGE and immunoblotted with antibodies to SMARCAL1 or RPA32. A coomassie stained gel was also prepared to document the amount of GST proteins added to the lysates. (B) Recombinant RPA, SMARCAL1, or both proteins were incubated with GST-WRN fragments bound to glutathione beads. After washing, bound proteins were separated by SDS-PAGE and immunoblotted with the indicated antibodies. (C) Flag immunoprecipitates from 293T cell lysates after transfection with Flag-SMARCAL1 wild-type (WT) or mutant expression vectors were separated by SDS-PAGE and immunoblotted with the indicated antibodies.</p

SMARCAL1 and WRN work independently to catalyze fork regression.

<p>Purified SMARCAL1 and WRN proteins were incubated with a ³²P-labeled model replication fork substrate capable of undergoing fork regression (see supplemental table S1). The substrate contains a 30-nucleotide ssDNA gap on the leading strand. RPA was pre-bound to the substrate prior to the addition of enzymes. (A) 500pM SMARCAL1 was used where indicated. The Ctl reactions lacked WRN protein to show the amount of spontaneous fork regression without SMARCAL1 or the amount of fork regression in the presence of only SMARCAL1. R764Q indicates reactions with the ATPase deficient SMARCAL1 R764Q mutant as a control. (B) Quantitation of the percent of product formed in each reaction. The amount of spontaneous branch migration in the absence of any added protein was subtracted from each sample.</p

Function of a Conserved Checkpoint Recruitment Domain in ATRIP Proteins▿

The ATR (ATM and Rad3-related) kinase is essential to maintain genomic integrity. ATR is recruited to DNA lesions in part through its association with ATR-interacting protein (ATRIP), which in turn interacts with the single-stranded DNA binding protein RPA (replication protein A). In this study, a conserved checkpoint protein recruitment domain (CRD) in ATRIP orthologs was identified by biochemical mapping of the RPA binding site in combination with nuclear magnetic resonance, mutagenesis, and computational modeling. Mutations in the CRD of the Saccharomyces cerevisiae ATRIP ortholog Ddc2 disrupt the Ddc2-RPA interaction, prevent proper localization of Ddc2 to DNA breaks, sensitize yeast to DNA-damaging agents, and partially compromise checkpoint signaling. These data demonstrate that the CRD is critical for localization and optimal DNA damage responses. However, the stimulation of ATR kinase activity by binding of topoisomerase binding protein 1 (TopBP1) to ATRIP-ATR can occur independently of the interaction of ATRIP with RPA. Our results support the idea of a multistep model for ATR activation that requires separable localization and activation functions of ATRIP