Mutational analysis of Brh2 reveals requirements for compensating mediator functions - PubMed (original) (raw)
Mutational analysis of Brh2 reveals requirements for compensating mediator functions
Milorad Kojic et al. Mol Microbiol. 2011 Jan.
Abstract
Brh2, a member of the BRCA2 family of proteins, governs homologous recombination in the fungus Ustilago maydis through interaction with Rad51. Brh2 serves at an early step in homologous recombination to mediate Rad51 nucleoprotein filament formation and also has the capability to function at a later step in recombination through its inherent DNA annealing activity. Rec2, a Rad51 paralogue, and Rad52 are additional components of the homologous recombination system, but the absence of either is less critical than Brh2 for operational activity. Here we tested a variety of mutant forms of Brh2 for activity in recombinational repair as measured by DNA repair proficiency. We found that a mutant of Brh2 deleted of the non-canonical DNA-binding domain within the N-terminal region is dependent upon the presence of Rad52 for DNA repair activity. We also determined that a motif first identified in human BRCA2 as important in binding DMC1 also contributes to DNA repair proficiency and cooperates with the BRC element in Rad51 binding.
© 2010 Blackwell Publishing Ltd.
Figures
Figure 1
Transposon scanning mutagenesis. A. Schematic representation of Brh2 with protein elements and domains as follows: BRC -- Rad51 interaction element; NBD -- N-terminal DNA binding domain; HD -- helix rich domain; OB1--OB module; OB2/tower- OB module with tower insert; CRE – Rad51 interaction region. Sites of 15 bp insertions, with corresponding translations, introduced after excision of the indicated transposons are shown. B. Mutant strains expressing the indicated Brh2 were grown to late log phase. Aliquots were adjusted to a density of 2 × 107 cells per ml, spotted on solid medium as serial 10-fold dilutions from left to right, and survival was determined after irradiation with UV at a dose of 120 J/m2.
Figure 2
Brh2 deletion mapping and domain swapping. A. Schematic representations of Brh2’s with various fragments deleted or replaced by DNA binding modules OB-A and OB-B from U. maydis RPA70. B. Mutant strains expressing the indicated Brh2 constructs were tested for survival after irradiation with UV at a dose of 120 J/m2.
Figure 3
Contribution of Phe(P/T)P and FTTP motifs to DNA repair activity. A. Schematic representation of Brh2 with mutated residues in BRC, Phe(P/T)P, and FTTP sequence motifs. brh2 strains expressing the indicated Brh2 point mutation constructs were tested for survival after irradiation with UV at a dose of 120 J/m2. B. Schematic representation of Brh2ΔC551 with Phe(P/T)P point mutations. brh2 expressing the indicated Brh2ΔC551 constructs were tested for survival. C. Schematic representation of Brh2 with Phe(P/T)P and CRE point mutations. Constructs were tested for survival.
Figure 4
Mutation in Brh2 Phe(P/T)P motif disturbs Rad51 binding. A. Schematic representation shows Brh2ΔC551 with mutated residues in BRC and Phe(P/T)P. Brh2ΔC551 and derivatives were tested for ability to form complexes with Rad51. Pulldowns of MBP-tagged Brh2ΔC551 variants plus associated Rad51 protein were performed as described in the experimental procedures. Left panel- preload samples; right panel-complexes eluted from amylose resin with maltose. Relative levels of Rad51 and Brh2ΔC551 were quantified by comparing the band intensity after staining. Averages of triplicates are presented with the standard error. Variants of Brh2ΔC551 with indicated mutations are as follows: lane a-wild type (wt); b-F294A T296A; c-F488A; d- F294A T296A F488A; e-no Brh2ΔC551 variant. B. Schematic representation shows Brh2ΔN359 with mutated residues F488A in Phe(P/T)P and W1052 in CRE. Pulldowns of MBP-tagged Brh2ΔN359 variants plus associated Rad51 protein from cleared E. coli extracts were performed as in experimental procedures. Left panel- preload samples; right panel-complexes eluted from amylose resin with maltose. Relative levels of Rad51 and Brh2ΔC551 were quantified by comparing the band intensity after ECL development. lane a-wild type (wt); b-F488A; c-W1052A; d-F488A W1052A; e-no Brh2ΔN359 variant.
Figure 5
DNA repair proficiency of Brh2 Phe(P/T)P and FTTP point mutations in the absence of Rec2 function. A. rec2 strains expressing the indicated Brh2 point mutation constructs were tested for survival after irradiation with UV at a dose of 120 J/m2. B Schematic representation of Rec2 (781 amino acids) RecA-related sequence and lysine residue K257 within the Walker A motif. rec2 expressing the indicated Rec2 K257R or K257A mutant constructs or co-expressing the Brh2 F488A Phe(P/T)P motif mutant and Rec2 point mutants were tested for survival. C. rec2 strains expressing Brh2ΔC551 with the indicated mutations were tested for survival after irradiation with gamma rays at a dose of 400 Gy.
Figure 6
Contribution of Rad52 domains to DNA repair proficiency of Bhr2 deleted of NBD. A. Schematic showing Brh2 deleted of NBD and Rad52 with its DNA binding domain (DBD) plus N- and C-terminal truncations. B. Survival of brh2 rad52 mutant strain expressing the indicated constructs after irradiation with UV at a dose of 120 J/m2.
Similar articles
- Brh2-Dss1 interplay enables properly controlled recombination in Ustilago maydis.
Kojic M, Zhou Q, Lisby M, Holloman WK. Kojic M, et al. Mol Cell Biol. 2005 Apr;25(7):2547-57. doi: 10.1128/MCB.25.7.2547-2557.2005. Mol Cell Biol. 2005. PMID: 15767662 Free PMC article. - Compensatory role for Rad52 during recombinational repair in Ustilago maydis.
Kojic M, Mao N, Zhou Q, Lisby M, Holloman WK. Kojic M, et al. Mol Microbiol. 2008 Mar;67(5):1156-68. doi: 10.1111/j.1365-2958.2008.06116.x. Epub 2008 Jan 15. Mol Microbiol. 2008. PMID: 18208529 - DNA binding, annealing, and strand exchange activities of Brh2 protein from Ustilago maydis.
Mazloum N, Zhou Q, Holloman WK. Mazloum N, et al. Biochemistry. 2007 Jun 19;46(24):7163-73. doi: 10.1021/bi700399m. Epub 2007 May 25. Biochemistry. 2007. PMID: 17523678 Free PMC article. - The homologous recombination system of Ustilago maydis.
Holloman WK, Schirawski J, Holliday R. Holloman WK, et al. Fungal Genet Biol. 2008 Aug;45 Suppl 1(Suppl 1):S31-9. doi: 10.1016/j.fgb.2008.04.006. Epub 2008 May 23. Fungal Genet Biol. 2008. PMID: 18502156 Free PMC article. Review. - Role of the human RAD51 protein in homologous recombination and double-stranded-break repair.
Baumann P, West SC. Baumann P, et al. Trends Biochem Sci. 1998 Jul;23(7):247-51. doi: 10.1016/s0968-0004(98)01232-8. Trends Biochem Sci. 1998. PMID: 9697414 Review.
Cited by
- DMC1 and RAD51 bind FxxA and FxPP motifs of BRCA2 via two separate interfaces.
Miron S, Legrand P, Dupaigne P, van Rossum-Fikkert SE, Ristic D, Majeed A, Kanaar R, Zinn-Justin S, Zelensky AN. Miron S, et al. Nucleic Acids Res. 2024 Jul 8;52(12):7337-7353. doi: 10.1093/nar/gkae452. Nucleic Acids Res. 2024. PMID: 38828772 Free PMC article. - DNA binding and RAD51 engagement by the BRCA2 C-terminus orchestrate DNA repair and replication fork preservation.
Kwon Y, Rösner H, Zhao W, Selemenakis P, He Z, Kawale AS, Katz JN, Rogers CM, Neal FE, Badamchi Shabestari A, Petrosius V, Singh AK, Joel MZ, Lu L, Holloway SP, Burma S, Mukherjee B, Hromas R, Mazin A, Wiese C, Sørensen CS, Sung P. Kwon Y, et al. Nat Commun. 2023 Jan 26;14(1):432. doi: 10.1038/s41467-023-36211-x. Nat Commun. 2023. PMID: 36702902 Free PMC article. - Loss of Cohesin Subunit Rec8 Switches Rad51 Mediator Dependence in Resistance to Formaldehyde Toxicity in Ustilago maydis.
Sutherland JH, Holloman WK. Sutherland JH, et al. Genetics. 2018 Oct;210(2):559-572. doi: 10.1534/genetics.118.301439. Epub 2018 Aug 6. Genetics. 2018. PMID: 30082279 Free PMC article. - Collaboration in the actions of Brh2 with resolving functions during DNA repair and replication stress in Ustilago maydis.
Kojic M, Milisavljevic M, Holloman WK. Kojic M, et al. DNA Repair (Amst). 2018 Mar;63:47-55. doi: 10.1016/j.dnarep.2018.01.010. Epub 2018 Feb 2. DNA Repair (Amst). 2018. PMID: 29414053 Free PMC article. - Dss1 Regulates Association of Brh2 with Rad51.
Zhou Q, Holloman WK. Zhou Q, et al. Biochemistry. 2017 Jul 5;56(26):3318-3327. doi: 10.1021/acs.biochem.7b00184. Epub 2017 Jun 26. Biochemistry. 2017. PMID: 28616972 Free PMC article.
References
- Budzowska M, Kanaar R. Mechanisms of dealing with DNA damage-induced replication problems. Cell Biochem Biophys. 2009;53:17–31. - PubMed
- Davies AA, Masson JY, McIlwraith MJ, Stasiak AZ, Stasiak A, Venkitaraman AR, West SC. Role of BRCA2 in control of the RAD51 recombination and DNA repair protein. Mol Cell. 2001;7:273–282. - PubMed
Publication types
MeSH terms
Substances
Grants and funding
- R01 GM079859-03/GM/NIGMS NIH HHS/United States
- R01 GM042482/GM/NIGMS NIH HHS/United States
- GM042482/GM/NIGMS NIH HHS/United States
- R01 GM042482-21/GM/NIGMS NIH HHS/United States
- GM079859/GM/NIGMS NIH HHS/United States
- R01 GM042482-20/GM/NIGMS NIH HHS/United States
- R01 GM079859-04/GM/NIGMS NIH HHS/United States
- R01 GM079859/GM/NIGMS NIH HHS/United States
LinkOut - more resources
Full Text Sources
Research Materials
Miscellaneous