The biochemistry of early meiotic recombination intermediates

Crickard, J. Brooks; Greene, Eric C.

doi:10.1080/15384101.2018.1553355

Cited by 27 publications

(32 citation statements)

References 109 publications

(160 reference statements)

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“…Both positive ( 1 ) and negative ( 2 , 7 , 8 , 9 , 10 ) controls grew and behaved as expected in a filter-based β-galactosidase assay: blue—positive, white—negative. Both full-length MCM9 ( 3 ) and the CTE only ( 4 ) showed blue color indicative of an interaction with RAD51. Mutation of the BRCv motif in both constructs ( 5 , 6 ) eliminated the positive blue color consistent with a disruption of the RAD51 interaction.…”

Section: Resultsmentioning

confidence: 99%

“…Homologous recombination (HR) of DNA involves multifaceted processes and pathways that respond to various types of DNA damage agents encountered during S/G2 phases of mitotic cells ( 1 , 2 ). Recombination occurring during meiosis can generate crossovers for genetic diversity and proper segregation in germline cells, utilizing many of the same enzymes ( 3 , 4 ). Therefore, HR is vital for genomic integrity and diversity required for organismal survival.…”

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confidence: 99%

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Motifs of the C-terminal domain of MCM9 direct localization to sites of mitomycin-C damage for RAD51 recruitment

McKinzey

Gomathinayagam

Griffin

et al. 2021

Journal of Biological Chemistry

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The MCM8/9 complex is implicated in aiding fork progression and facilitating homologous recombination (HR) in response to several DNA damage agents. MCM9 itself is an outlier within the MCM family containing a long C-terminal extension (CTE) comprising 42% of the total length, but with no known functional components and high predicted disorder. In this report, we identify and characterize two unique motifs within the primarily unstructured CTE that are required for localization of MCM8/9 to sites of mitomycin C (MMC)-induced DNA damage. First, an unconventional “bipartite-like” nuclear localization (NLS) motif consisting of two positively charged amino acid stretches separated by a long intervening sequence is required for the nuclear import of both MCM8 and MCM9. Second, a variant of the BRC motif (BRCv) similar to that found in other HR helicases is necessary for localization to sites of MMC damage. The MCM9-BRCv directly interacts with and recruits RAD51 downstream to MMC-induced damage to aid in DNA repair. Patient lymphocytes devoid of functional MCM9 and discrete MCM9 knockout cells have a significantly impaired ability to form RAD51 foci after MMC treatment. Therefore, the disordered CTE in MCM9 is functionally important in promoting MCM8/9 activity and in recruiting downstream interactors; thus, requiring full-length MCM9 for proper DNA repair.

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Motifs of the C-terminal domain of MCM9 direct localization to sites of mitomycin-C damage for RAD51 recruitment

McKinzey

Gomathinayagam

Griffin

et al. 2021

Journal of Biological Chemistry

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show abstract

“…Homologous recombination (HR) of DNA involves multifaceted processes and pathways that respond to various types of DNA damage agents encountered during S/G2-phases of mitotic cells (Quinet et al 2017;Wright et al 2018). Recombination occurring during meiosis can generate crossovers for genetic diversity and proper segregation in germline cells, utilizing many of the same enzymes (Hunter 2015;Crickard et al 2018). Therefore, HR is vital for genomic integrity and diversity required for organismal survival.…”

Section: Introductionmentioning

confidence: 99%

Motifs of the C-terminal Domain of MCM9 Direct Localization to Sites of Mitomycin-C Damage for RAD51 Recruitment

McKinzey

Gomanthinayagam

Griffin

et al. 2020

Preprint

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The MCM8/9 complex is implicated in aiding fork progression and facilitating homologous recombination (HR) in response to several DNA damage agents. MCM9 itself is an outlier within the MCM family containing a long C-terminal extension (CTE) comprising 42% of the total length, but with no known functional components and high predicted disorder. In this report, we identify and characterize two unique motifs within the primarily unstructured CTE that are required for localization of MCM8/9 to sites of mitomycin C (MMC) induced DNA damage. First, an unconventional ‘bipartite-like’ nuclear localization (NLS) motif consisting of two positively charged amino acid stretches separated by a long intervening sequence is required for the nuclear import of both MCM8 and MCM9. Second, a variant of the BRC motif (BRCv), similar to that found in other HR helicases, is necessary for localization to sites of MMC damage. The MCM9-BRCv directly interacts with and recruits RAD51 downstream to MMC-induced damage to aid in DNA repair. Patient lymphocytes devoid of functional MCM9 and discrete MCM9 knockout cells have a significantly impaired ability to form RAD51 foci after MMC treatment. Therefore, the disordered CTE in MCM9 is functionally important in promoting MCM8/9 activity and in recruiting downstream interactors; thus, requiring full length MCM9 for proper DNA repair.

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“…Once homology is located, recombinasedriven DNA pairing forms a displacement loop (D-loop) for the subsequent strand-exchange process. Thus, the assembly of nucleoprotein filament is the first committed step in the recombination pathway and has been subject to tight regulation (1,2).…”

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confidence: 99%

“…In addition to the similarities between Dmc1 and Rad51, differences exist. Dmc1 is present only in meiosis while Rad51 is expressed in both meiotic and mitotic cells (2,8). Dmc1 is shown to have a better tolerance in mismatch during meiotic recombination (6,(11)(12)(13)(14).…”

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confidence: 99%

Rad51 facilitates filament assembly of meiosis-specific Dmc1 recombinase

Lan

Lin

Kao

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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Dmc1 recombinases are essential to homologous recombination in meiosis. Here, we studied the kinetics of the nucleoprotein filament assembly ofSaccharomyces cerevisiaeDmc1 using single-molecule tethered particle motion experiments and in vitro biochemical assay. ScDmc1 nucleoprotein filaments are less stable than the ScRad51 ones because of the kinetically much reduced nucleation step. The lower nucleation rate of ScDmc1 results from its lower single-stranded DNA (ssDNA) affinity, compared to that of ScRad51. Surprisingly, ScDmc1 nucleates mostly on the DNA structure containing the single-stranded and duplex DNA junction with the allowed extension in the 5′-to-3′ polarity, while ScRad51 nucleation depends strongly on ssDNA lengths. This nucleation preference is also conserved for mammalian RAD51 and DMC1. In addition, ScDmc1 nucleation can be stimulated by short ScRad51 patches, but not by EcRecA ones. Pull-down experiments also confirm the physical interactions of ScDmc1 with ScRad51 in solution, but not with EcRecA. Our results are consistent with a model that Dmc1 nucleation can be facilitated by a structural component (such as DNA junction and protein–protein interaction) and DNA polarity. They provide direct evidence of how Rad51 is required for meiotic recombination and highlight a regulation strategy in Dmc1 nucleoprotein filament assembly.

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The biochemistry of early meiotic recombination intermediates

Cited by 27 publications

References 109 publications

Motifs of the C-terminal domain of MCM9 direct localization to sites of mitomycin-C damage for RAD51 recruitment

Motifs of the C-terminal domain of MCM9 direct localization to sites of mitomycin-C damage for RAD51 recruitment

Motifs of the C-terminal Domain of MCM9 Direct Localization to Sites of Mitomycin-C Damage for RAD51 Recruitment

Rad51 facilitates filament assembly of meiosis-specific Dmc1 recombinase

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