Limits...
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.

Show MeSH

Related in: MedlinePlus

RAD9 localizes to meiotic chromosomes during early prophase I and colocalizes with a subset of RAD51 foci.A–C. Meiotic chromosome spreads from control animals were stained for RAD9 and SYCP3. In wild-type adult males, RAD9 localized along the synaptonemal complex of synapsed and unsynapsed chromosomes during zygotene (A) and pachytene (B, C). RAD9 localized to autosomes and the sex chromosomes in early pachytene (B), was confined primarily to the X chromosome by mid-late pachytene (C), and was absent by diplotene. D. Meiotic chromosome spreads from control animals were stained for RAD9 and RAD51. RAD9 colocalized with a subset of RAD51 foci, particularly along the X chromosome in pachytene-like nuclei.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3585019&req=5

pgen-1003320-g007: RAD9 localizes to meiotic chromosomes during early prophase I and colocalizes with a subset of RAD51 foci.A–C. Meiotic chromosome spreads from control animals were stained for RAD9 and SYCP3. In wild-type adult males, RAD9 localized along the synaptonemal complex of synapsed and unsynapsed chromosomes during zygotene (A) and pachytene (B, C). RAD9 localized to autosomes and the sex chromosomes in early pachytene (B), was confined primarily to the X chromosome by mid-late pachytene (C), and was absent by diplotene. D. Meiotic chromosome spreads from control animals were stained for RAD9 and RAD51. RAD9 colocalized with a subset of RAD51 foci, particularly along the X chromosome in pachytene-like nuclei.

Mentions: Since Hus1 disruption resulted in chromosomal defects without affecting TOPBP1 localization, we next analyzed RAD9 localization to meiotic chromosomes to gain insights into the normal functions of 9-1-1. As shown in Figure 7A–7C, RAD9 localized to meiotic chromosome cores during prophase I, with many foci on both unsynapsed and synapsed chromosomes during zygotene stage, fewer autosomal foci and prominent X chromosome foci in early pachytene, and even fewer foci by mid- to late-pachytene stage when most staining was confined to bright foci along the X chromosome core. By diplotene stage, RAD9 foci were not detectable on meiotic chromosomes. Thus, the RAD9 subunit of 9-1-1 is appropriately positioned for a potential role in genome maintenance during meiosis.


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)

RAD9 localizes to meiotic chromosomes during early prophase I and colocalizes with a subset of RAD51 foci.A–C. Meiotic chromosome spreads from control animals were stained for RAD9 and SYCP3. In wild-type adult males, RAD9 localized along the synaptonemal complex of synapsed and unsynapsed chromosomes during zygotene (A) and pachytene (B, C). RAD9 localized to autosomes and the sex chromosomes in early pachytene (B), was confined primarily to the X chromosome by mid-late pachytene (C), and was absent by diplotene. D. Meiotic chromosome spreads from control animals were stained for RAD9 and RAD51. RAD9 colocalized with a subset of RAD51 foci, particularly along the X chromosome in pachytene-like nuclei.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1003320-g007: RAD9 localizes to meiotic chromosomes during early prophase I and colocalizes with a subset of RAD51 foci.A–C. Meiotic chromosome spreads from control animals were stained for RAD9 and SYCP3. In wild-type adult males, RAD9 localized along the synaptonemal complex of synapsed and unsynapsed chromosomes during zygotene (A) and pachytene (B, C). RAD9 localized to autosomes and the sex chromosomes in early pachytene (B), was confined primarily to the X chromosome by mid-late pachytene (C), and was absent by diplotene. D. Meiotic chromosome spreads from control animals were stained for RAD9 and RAD51. RAD9 colocalized with a subset of RAD51 foci, particularly along the X chromosome in pachytene-like nuclei.
Mentions: Since Hus1 disruption resulted in chromosomal defects without affecting TOPBP1 localization, we next analyzed RAD9 localization to meiotic chromosomes to gain insights into the normal functions of 9-1-1. As shown in Figure 7A–7C, RAD9 localized to meiotic chromosome cores during prophase I, with many foci on both unsynapsed and synapsed chromosomes during zygotene stage, fewer autosomal foci and prominent X chromosome foci in early pachytene, and even fewer foci by mid- to late-pachytene stage when most staining was confined to bright foci along the X chromosome core. By diplotene stage, RAD9 foci were not detectable on meiotic chromosomes. Thus, the RAD9 subunit of 9-1-1 is appropriately positioned for a potential role in genome maintenance during meiosis.

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