title: A short carboxyl-terminal tail is required for single-stranded DNA binding, higher-order structural organization, and stability of the mitochondrial single-stranded annealing protein Mgm101.
abstract: Mgm101 is a Rad52-type single-stranded annealing protein (SSAP) required for mitochondrial DNA (mtDNA) repair and maintenance. Structurally, Mgm101 forms large oligomeric rings. Here we determine the function(s) of a 32-amino acid carboxyl-terminal tail (Mgm101(238-269)) conserved in the Mgm101 family of proteins. Mutagenic analysis shows that Lys-253, Trp-257, Arg-259, and Tyr-268 are essential for mtDNA maintenance. Mutations in Lys-251, Arg-252, Lys-260, and Tyr-266 affect mtDNA stability at 37°C and under oxidative stress. The Y268A mutation severely affects single-stranded DNA (ssDNA) binding without altering the ring structure. Mutations in the Lys-251-Arg-252-Lys-253 positive triad also affect ssDNA binding. Moreover, the C-tail alone is sufficient to mediate ssDNA binding. Finally, we find that the W257A and R259A mutations dramatically affect the conformation and oligomeric state of Mgm101. These structural alterations correlate with protein degradation in vivo. The data thus indicate that the C-tail of Mgm101, likely displayed on the ring surface, is required for ssDNA binding, higher-order structural organization, and protein stability. We speculate that an initial electrostatic and base-stacking interaction with ssDNA could remodel ring organization. This may facilitate the formation of nucleoprotein filaments competent for mtDNA repair. These findings could have broad implications for understanding how SSAPs promote DNA repair and genome maintenance.
authors.affiliation: Department of Biochemistry and Molecular Biology, State University of New York Upstate Medical University, Syracuse, NY 13210, USA.
authors.forename: MacMillan
authors.initials: M
authors.lastname: Mbantenkhu
authors.forename: Sara
authors.initials: S
authors.lastname: Wierzbicki
authors.forename: Xiaowen
authors.initials: X
authors.lastname: Wang
authors.forename: Shangdong
authors.initials: S
authors.lastname: Guo
authors.forename: Stephan
authors.initials: S
authors.lastname: Wilkens
authors.forename: Xin Jie
authors.initials: XJ
authors.lastname: Chen
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D017124:Conserved Sequence; D004272:DNA, Mitochondrial / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005816:Genetic Complementation Test; D024101:Mitochondrial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D055550:Protein Stability; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D004272:DNA, Mitochondrial; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; C082519:MGM101 protein, S cerevisiae; D024101:Mitochondrial Proteins; D029701:Saccharomyces cerevisiae Proteins
references.citation: Nat Protoc. 2009;4(3):363-71
references.pmid: 19247286
references.citation: Mol Cell. 2002 Aug;10(2):359-71
references.pmid: 12191481
references.citation: Nature. 1998 Jan 22;391(6665):404-7
references.pmid: 9450759
references.citation: Microbiol Rev. 1994 Sep;58(3):401-65
references.pmid: 7968921
references.citation: EMBO J. 1995 Aug 15;14(16):4090-101
references.pmid: 7664749
references.citation: Curr Biol. 2000 Mar 23;10(6):337-40
references.pmid: 10744977
references.citation: Curr Biol. 2008 Aug 5;18(15):1142-6
references.pmid: 18656357
references.citation: Proc Natl Acad Sci U S A. 2001 Jul 17;98(15):8306-11
references.pmid: 11459968
references.citation: J Mol Biol. 1983 Dec 25;171(4):401-18
references.pmid: 6607360
references.citation: Annu Rev Biochem. 2008;77:229-57
references.pmid: 18275380
references.citation: J Biol Chem. 2008 May 2;283(18):12166-74
references.pmid: 18310075
references.citation: Proc Natl Acad Sci U S A. 1996 Oct 1;93(20):10729-34
references.pmid: 8855248
references.citation: Microbiol Mol Biol Rev. 2002 Dec;66(4):630-70, table of contents
references.pmid: 12456786
references.citation: EMBO J. 2002 Sep 2;21(17):4730-40
references.pmid: 12198175
references.citation: Plant Cell. 2011 Dec;23(12):4266-79
references.pmid: 22202891
references.citation: Mol Microbiol. 2008 Apr;68(1):66-74
references.pmid: 18333884
references.citation: Genes Cells. 1998 Mar;3(3):145-56
references.pmid: 9619627
references.citation: EMBO J. 2006 Nov 29;25(23):5539-48
references.pmid: 17093500
references.citation: J Mol Biol. 2009 Aug 21;391(3):586-98
references.pmid: 19527729
references.citation: Genetics. 2001 Oct;159(2):515-25
references.pmid: 11606529
references.citation: J Biol Chem. 2009 Nov 27;284(48):33275-84
references.pmid: 19812039
references.citation: Mitochondrion. 2011 Jul;11(4):575-86
references.pmid: 21406253
references.citation: FEMS Yeast Res. 2007 Jan;7(1):131-40
references.pmid: 17311591
references.citation: Proc Natl Acad Sci U S A. 2000 Jul 5;97(14):7772-7
references.pmid: 10869431
references.citation: J Mol Biol. 2008 Dec 31;384(5):1098-105
references.pmid: 18983848
references.citation: Genes Dev. 1996 Aug 15;10(16):2025-37
references.pmid: 8769646
references.citation: Proc Natl Acad Sci U S A. 1998 May 26;95(11):6049-54
references.pmid: 9600915
references.citation: Nucleic Acids Res. 2010 May;38(9):2917-30
references.pmid: 20081207
references.citation: Plant J. 2012 Nov;72(3):423-35
references.pmid: 22762281
references.citation: Genetics. 2002 Apr;160(4):1389-400
references.pmid: 11973295
references.citation: Genetics. 2012 Mar;190(3):951-64
references.pmid: 22214610
references.citation: Proc Natl Acad Sci U S A. 2002 Oct 15;99(21):13492-7
references.pmid: 12370410
references.citation: BMC Genomics. 2002 Mar 21;3:8
references.pmid: 11914131
references.citation: Nucleic Acids Res. 2010 Jul;38(12):3952-62
references.pmid: 20194117
references.citation: J Cell Biol. 1999 Apr 19;145(2):291-304
references.pmid: 10209025
references.citation: Proc Natl Acad Sci U S A. 1999 Apr 13;96(8):4279-84
references.pmid: 10200253
references.citation: Annu Rev Biophys Biomol Struct. 2003;32:115-33
references.pmid: 12598368
references.citation: J Mol Biol. 1970 Dec 28;54(3):417-40
references.pmid: 4923667
references.citation: Proc Natl Acad Sci U S A. 2008 Dec 23;105(51):20274-9
references.pmid: 19074292
references.citation: PLoS One. 2013;8(2):e56465
references.pmid: 23418572
references.citation: J Biol Chem. 2001 Sep 14;276(37):35201-8
references.pmid: 11454867
references.citation: Genes Dev. 1993 Sep;7(9):1755-65
references.pmid: 8370524
references.citation: J Biol Chem. 2006 Jun 2;281(22):15441-9
references.pmid: 16565518
references.citation: Virology. 1984 Apr 15;134(1):148-60
references.pmid: 6369766
references.citation: Mol Cell Biol. 1998 Jul;18(7):4400-6
references.pmid: 9632824
references.citation: Nucleic Acids Res. 1993 Jul 25;21(15):3473-7
references.pmid: 8346025
references.citation: DNA Repair (Amst). 2007 Jan 4;6(1):27-37
references.pmid: 16987715
references.citation: J Biol Chem. 2012 Oct 26;287(44):37259-68
references.pmid: 22948312
references.citation: Cell. 1992 May 1;69(3):457-70
references.pmid: 1581961
references.citation: Genetics. 1997 Nov;147(3):961-77
references.pmid: 9383045
references.citation: J Mol Biol. 2005 Jan 14;345(2):239-49
references.pmid: 15571718
references.citation: J Biol Chem. 2011 Dec 9;286(49):42360-42370
references.pmid: 22027892
references.citation: Mol Gen Genet. 1996 Oct 28;252(6):746-50
references.pmid: 8917319