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Conditional inactivation of the DNA damage response gene Hus1 in mouse testis reveals separable roles for components of the RAD9-RAD1-HUS1 complex in meiotic chromosome maintenance.

Lyndaker AM, Lim PX, Mleczko JM, Diggins CE, Holloway JK, Holmes RJ, Kan R, Schlafer DH, Freire R, Cohen PE, Weiss RS - PLoS Genet. (2013)

Bottom Line: Hus1 loss in testicular germ cells resulted in meiotic defects, germ cell depletion, and severely compromised fertility.Interestingly, RAD1 had a broader distribution that only partially overlapped with RAD9, and localization of both RAD1 and the ATR activator TOPBP1 to the XY body and to unsynapsed autosomes was intact in Hus1 conditional knockouts.We conclude that mammalian HUS1 acts as a component of the canonical 9-1-1 complex during meiotic prophase I to promote DSB repair and further propose that RAD1 and TOPBP1 respond to unsynapsed chromatin through an alternative mechanism that does not require RAD9 or HUS1.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, Cornell University, Ithaca, New York, USA.

ABSTRACT
The RAD9-RAD1-HUS1 (9-1-1) complex is a heterotrimeric PCNA-like clamp that responds to DNA damage in somatic cells by promoting DNA repair as well as ATR-dependent DNA damage checkpoint signaling. In yeast, worms, and flies, the 9-1-1 complex is also required for meiotic checkpoint function and efficient completion of meiotic recombination; however, since Rad9, Rad1, and Hus1 are essential genes in mammals, little is known about their functions in mammalian germ cells. In this study, we assessed the meiotic functions of 9-1-1 by analyzing mice with germ cell-specific deletion of Hus1 as well as by examining the localization of RAD9 and RAD1 on meiotic chromosomes during prophase I. Hus1 loss in testicular germ cells resulted in meiotic defects, germ cell depletion, and severely compromised fertility. Hus1-deficient primary spermatocytes exhibited persistent autosomal γH2AX and RAD51 staining indicative of unrepaired meiotic DSBs, synapsis defects, an extended XY body domain often encompassing partial or whole autosomes, and an increase in structural chromosome abnormalities such as end-to-end X chromosome-autosome fusions and ruptures in the synaptonemal complex. Most of these aberrations persisted in diplotene-stage spermatocytes. Consistent with a role for the 9-1-1 complex in meiotic DSB repair, RAD9 localized to punctate, RAD51-containing foci on meiotic chromosomes in a Hus1-dependent manner. Interestingly, RAD1 had a broader distribution that only partially overlapped with RAD9, and localization of both RAD1 and the ATR activator TOPBP1 to the XY body and to unsynapsed autosomes was intact in Hus1 conditional knockouts. We conclude that mammalian HUS1 acts as a component of the canonical 9-1-1 complex during meiotic prophase I to promote DSB repair and further propose that RAD1 and TOPBP1 respond to unsynapsed chromatin through an alternative mechanism that does not require RAD9 or HUS1.

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RAD51 foci persist in late pachytene and diplotene spermatocytes in the absence of Hus1.A–E. Immunofluorescence staining for RAD51/DMC1 from zygotene through diplotene stage of meiosis in control (Cre-negative Hus1flox/Δ1) and Stra8-Cre Hus1 CKO males. Arrows indicate persistent foci, and asterisks indicate symmetrical foci located on either side of chromosomal cores. F–I. Quantification of RAD51 foci at the indicated stages. ** indicates p<0.01; * indicates p<0.05.
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pgen-1003320-g004: RAD51 foci persist in late pachytene and diplotene spermatocytes in the absence of Hus1.A–E. Immunofluorescence staining for RAD51/DMC1 from zygotene through diplotene stage of meiosis in control (Cre-negative Hus1flox/Δ1) and Stra8-Cre Hus1 CKO males. Arrows indicate persistent foci, and asterisks indicate symmetrical foci located on either side of chromosomal cores. F–I. Quantification of RAD51 foci at the indicated stages. ** indicates p<0.01; * indicates p<0.05.

Mentions: Since increased numbers of γH2AX foci were present on Hus1 CKO meiotic chromosomes, we further investigated whether the RAD51 strand exchange protein accumulated normally on Hus1-deficient chromosomes. During the early stages of meiotic DSB repair in zygotene and early pachytene spermatocytes, RAD51 properly localized to punctate structures along meiotic chromosome cores in the absence of Hus1 (Figure 4A). By late pachytene, when most RAD51 foci are normally lost from autosomal and XY chromosome cores, all Hus1 CKO spermatocytes exhibited persistent localization of RAD51 to the autosomes as well as the X chromosome, indicative of a DSB repair defect (Figure 4C). These foci persisted into diplotene as well (Figure 4D), demonstrating that nuclei with unrepaired DSBs were also able to progress beyond the pachytene stage. RAD51 focus numbers were quantified at each of these stages (Figure 4F–4I), and the number of foci in Hus1 CKO nuclei was significantly increased in late pachytene and diplotene. While the number of persistent RAD51 foci per nucleus was modest, the total number of nuclei with at least one RAD51 focus was high in Hus1 CKOs, at 100% of Hus1 CKO nuclei versus 52% of controls in late pachytene, and 73% of Hus1 CKOs versus 17% of controls in diplotene (Figure 4). Further, the reduction in the frequency of cells with RAD51 foci from 100% to 73% of Hus1 CKO nuclei from late pachytene to diplotene could indicate that Hus1 mutants can repair DSBs, albeit inefficiently in some cases, such that repair is significantly delayed. We conclude that HUS1 is critical for efficient completion of DNA repair at a subset of meiotic DSBs.


Conditional inactivation of the DNA damage response gene Hus1 in mouse testis reveals separable roles for components of the RAD9-RAD1-HUS1 complex in meiotic chromosome maintenance.

Lyndaker AM, Lim PX, Mleczko JM, Diggins CE, Holloway JK, Holmes RJ, Kan R, Schlafer DH, Freire R, Cohen PE, Weiss RS - PLoS Genet. (2013)

RAD51 foci persist in late pachytene and diplotene spermatocytes in the absence of Hus1.A–E. Immunofluorescence staining for RAD51/DMC1 from zygotene through diplotene stage of meiosis in control (Cre-negative Hus1flox/Δ1) and Stra8-Cre Hus1 CKO males. Arrows indicate persistent foci, and asterisks indicate symmetrical foci located on either side of chromosomal cores. F–I. Quantification of RAD51 foci at the indicated stages. ** indicates p<0.01; * indicates p<0.05.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3585019&req=5

pgen-1003320-g004: RAD51 foci persist in late pachytene and diplotene spermatocytes in the absence of Hus1.A–E. Immunofluorescence staining for RAD51/DMC1 from zygotene through diplotene stage of meiosis in control (Cre-negative Hus1flox/Δ1) and Stra8-Cre Hus1 CKO males. Arrows indicate persistent foci, and asterisks indicate symmetrical foci located on either side of chromosomal cores. F–I. Quantification of RAD51 foci at the indicated stages. ** indicates p<0.01; * indicates p<0.05.
Mentions: Since increased numbers of γH2AX foci were present on Hus1 CKO meiotic chromosomes, we further investigated whether the RAD51 strand exchange protein accumulated normally on Hus1-deficient chromosomes. During the early stages of meiotic DSB repair in zygotene and early pachytene spermatocytes, RAD51 properly localized to punctate structures along meiotic chromosome cores in the absence of Hus1 (Figure 4A). By late pachytene, when most RAD51 foci are normally lost from autosomal and XY chromosome cores, all Hus1 CKO spermatocytes exhibited persistent localization of RAD51 to the autosomes as well as the X chromosome, indicative of a DSB repair defect (Figure 4C). These foci persisted into diplotene as well (Figure 4D), demonstrating that nuclei with unrepaired DSBs were also able to progress beyond the pachytene stage. RAD51 focus numbers were quantified at each of these stages (Figure 4F–4I), and the number of foci in Hus1 CKO nuclei was significantly increased in late pachytene and diplotene. While the number of persistent RAD51 foci per nucleus was modest, the total number of nuclei with at least one RAD51 focus was high in Hus1 CKOs, at 100% of Hus1 CKO nuclei versus 52% of controls in late pachytene, and 73% of Hus1 CKOs versus 17% of controls in diplotene (Figure 4). Further, the reduction in the frequency of cells with RAD51 foci from 100% to 73% of Hus1 CKO nuclei from late pachytene to diplotene could indicate that Hus1 mutants can repair DSBs, albeit inefficiently in some cases, such that repair is significantly delayed. We conclude that HUS1 is critical for efficient completion of DNA repair at a subset of meiotic DSBs.

Bottom Line: Hus1 loss in testicular germ cells resulted in meiotic defects, germ cell depletion, and severely compromised fertility.Interestingly, RAD1 had a broader distribution that only partially overlapped with RAD9, and localization of both RAD1 and the ATR activator TOPBP1 to the XY body and to unsynapsed autosomes was intact in Hus1 conditional knockouts.We conclude that mammalian HUS1 acts as a component of the canonical 9-1-1 complex during meiotic prophase I to promote DSB repair and further propose that RAD1 and TOPBP1 respond to unsynapsed chromatin through an alternative mechanism that does not require RAD9 or HUS1.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, Cornell University, Ithaca, New York, USA.

ABSTRACT
The RAD9-RAD1-HUS1 (9-1-1) complex is a heterotrimeric PCNA-like clamp that responds to DNA damage in somatic cells by promoting DNA repair as well as ATR-dependent DNA damage checkpoint signaling. In yeast, worms, and flies, the 9-1-1 complex is also required for meiotic checkpoint function and efficient completion of meiotic recombination; however, since Rad9, Rad1, and Hus1 are essential genes in mammals, little is known about their functions in mammalian germ cells. In this study, we assessed the meiotic functions of 9-1-1 by analyzing mice with germ cell-specific deletion of Hus1 as well as by examining the localization of RAD9 and RAD1 on meiotic chromosomes during prophase I. Hus1 loss in testicular germ cells resulted in meiotic defects, germ cell depletion, and severely compromised fertility. Hus1-deficient primary spermatocytes exhibited persistent autosomal γH2AX and RAD51 staining indicative of unrepaired meiotic DSBs, synapsis defects, an extended XY body domain often encompassing partial or whole autosomes, and an increase in structural chromosome abnormalities such as end-to-end X chromosome-autosome fusions and ruptures in the synaptonemal complex. Most of these aberrations persisted in diplotene-stage spermatocytes. Consistent with a role for the 9-1-1 complex in meiotic DSB repair, RAD9 localized to punctate, RAD51-containing foci on meiotic chromosomes in a Hus1-dependent manner. Interestingly, RAD1 had a broader distribution that only partially overlapped with RAD9, and localization of both RAD1 and the ATR activator TOPBP1 to the XY body and to unsynapsed autosomes was intact in Hus1 conditional knockouts. We conclude that mammalian HUS1 acts as a component of the canonical 9-1-1 complex during meiotic prophase I to promote DSB repair and further propose that RAD1 and TOPBP1 respond to unsynapsed chromatin through an alternative mechanism that does not require RAD9 or HUS1.

Show MeSH
Related in: MedlinePlus