During meiotic prophase in male mammals, the X and Y chromosomes are incorporated in the XY body. This heterochromatic body is transcriptionally silenced and marked by increased ubiquitination of histone H2A. This led us to investigate the relationship between histone H2A ubiquitination and chromatin silencing in more detail. First, we found that ubiquitinated H2A also marks the silenced X chromosome of the Barr body in female somatic cells. Next, we studied a possible relationship between H2A ubiquitination, chromatin silencing, and unpaired chromatin in meiotic prophase. The mouse models used carry an unpaired autosomal region in male meiosis or unpaired X and Y chromosomes in female meiosis. We show that ubiquitinated histone H2A is associated with transcriptional silencing of large chromatin regions. This silencing in mammalian meiotic prophase cells concerns unpaired chromatin regions and resembles a phenomenon described for the fungus Neurospora crassa and named meiotic silencing by unpaired DNA.Chromatin remodeling is at the basis of control of cellspecific gene expression, cell determination, and differentiation. The nucleosome units of chromatin consist of two each of the histones H2A, H2B, H3, and H4. The N-terminal ends of these core histones extend from the nucleosome and can undergo posttranslational covalent modifications, such as methylation, acetylation, phosphorylation, and ADP-ribosylation of specific amino acid residues. Together, these modifications constitute the so-called histone code (45). Interaction of other nuclear proteins with chromatin is dependent on the histone code at specific chromatin regions and determines local chromatin structure, which can be open or closed. A remarkable component of the histone code is ubiquitination of C-terminal lysine residues of histones H2A and H2B. Ubiquitin, a protein of 7 kDa, can be attached to lysine residues of a specific protein substrate through the action of a multienzyme complex containing ubiquitin-activating (E1), ubiquitin-conjugating (E2), and ubiquitin ligase (E3) enzymes. Polyubiquitination can target proteins for degradation by the proteasome (34, 35). Monoubiquitination of histones, however, is a stable modification that does not decrease the half-life of the target histone (56).In the yeast Saccharomyces cerevisiae, histone H2A ubiquitination is not required for cell growth or sporulation (47), but histone H2B ubiquitination is an essential mechanism involved in sporulation (37). Most importantly, it has been shown that ubiquitination of H2B by the ubiquitin-conjugating enzyme RAD6, interacting with the ubiquitin ligase BRE1, is a prerequisite for dimethylation of histone H3 at lysine residues 4 and 79 (5,12,37,46). This mechanism is thought to be associated with potentiation of gene activation. It is not known whether this "trans-histone" mechanism is conserved between yeast and mammals. RAD6 shows marked evolutionary conservation. The two mammalian homologs of yeast RAD6, Hr6a/Ube2a and Hr6b/Ube2b, both show approximately 70% amino...

Homologous recombination is a versatile DNA damage repair pathway requiring Rad51 and Rad54. Here we show that a mammalian Rad54 paralog, Rad54B, displays physical and functional interactions with Rad51 and DNA that are similar to those of Rad54. While ablation of Rad54 in mouse embryonic stem (ES) cells leads to a mild reduction in homologous recombination efficiency, the absence of Rad54B has little effect. However, the absence of both Rad54 and Rad54B dramatically reduces homologous recombination efficiency. Furthermore, we show that Rad54B protects ES cells from ionizing radiation and the interstrand DNA cross-linking agent mitomycin C. Interestingly, at the ES cell level the paralogs do not display an additive or synergic interaction with respect to mitomycin C sensitivity, yet animals lacking both Rad54 and Rad54B are dramatically sensitized to mitomycin C compared to either single mutant. This suggests that the paralogs possibly function in a tissue-specific manner. Finally, we show that Rad54, but not Rad54B, is needed for a normal distribution of Rad51 on meiotic chromosomes. Thus, even though the paralogs have similar biochemical properties, genetic analysis in mice uncovered their nonoverlapping roles.DNA double-strand breaks (DSBs) are among a plethora of lesions that threaten the integrity of the genome. If not properly processed, DSBs can lead to cell cycle arrest or illegitimate DNA rearrangements such as translocations, inversions, or deletions. These rearrangements can contribute to cell dysfunction, cell death, or carcinogenesis (22). DSBs can arise through the action of exogenous DNA-damaging agents, but they also arise from endogenous sources, such as oxidative DNA damage and as a consequence of DNA replication (10,22). Homologous recombination is a major DNA repair pathway by which DSBs are repaired. Homologous recombination is generally a precise way of resolving DSBs, because it uses homologous sequence, usually provided on the sister chromatid, as a repair template (54).Homologous recombination is a complex process requiring a number of proteins of the RAD52 epistasis group, including Rad51 and Rad54. Rad51 is the key player in this process because it is critical for homology recognition and performs strand exchange between recombining DNA molecules. A pivotal intermediate in these reactions is the Rad51 nucleoprotein filament. This forms when Rad51 polymerizes on singlestranded DNA that results from DNA damage processing (54). Rad54 is an important accessory factor for Rad51 (56). A number of biochemical characteristics of Rad54 have been well defined for different species ranging from yeasts to humans (8,18,24,31,37,38,42,47,48,53,55,59). Rad54 is a doublestranded-DNA-dependent ATPase that can translocate on DNA, thereby affecting DNA topology. Biochemically, Rad54 has been implicated in participation in multiple steps of homologous recombination. It can stabilize the Rad51 nucleoprotein filament in an early stage of recombination (30). At a subsequent stage it can promote chromatin rem...

During meiotic prophase in male mammals, the heterologous X and Y chromosomes remain largely unsynapsed, and meiotic sex chromosome inactivation (MSCI) leads to formation of the transcriptionally silenced XY body. In birds, the heterogametic sex is female, carrying Z and W chromosomes (ZW), whereas males have the homogametic ZZ constitution. During chicken oogenesis, the heterologous ZW pair reaches a state of complete heterologous synapsis, and this might enable maintenance of transcription of Z- and W chromosomal genes during meiotic prophase. Herein, we show that the ZW pair is transiently silenced, from early pachytene to early diplotene using immunocytochemistry and gene expression analyses. We propose that ZW inactivation is most likely achieved via spreading of heterochromatin from the W on the Z chromosome. Also, persistent meiotic DNA double-strand breaks (DSBs) may contribute to silencing of Z. Surprisingly, γH2AX, a marker of DSBs, and also the earliest histone modification that is associated with XY body formation in mammalian and marsupial spermatocytes, does not cover the ZW during the synapsed stage. However, when the ZW pair starts to desynapse, a second wave of γH2AX accumulates on the unsynapsed regions of Z, which also show a reappearance of the DSB repair protein RAD51. This indicates that repair of meiotic DSBs on the heterologous part of Z is postponed until late pachytene/diplotene, possibly to avoid recombination with regions on the heterologously synapsed W chromosome. Two days after entering diplotene, the Z looses γH2AX and shows reactivation. This is the first report of meiotic sex chromosome inactivation in a species with female heterogamety, providing evidence that this mechanism is not specific to spermatogenesis. It also indicates the presence of an evolutionary force that drives meiotic sex chromosome inactivation independent of the final achievement of synapsis.

The Saccharomyces cerevisiae RAD6 protein is required for a surprising diversity of cellular processes, including sporulation and replicational damage bypass of DNA lesions. In mammals, two RAD6-related genes, HR6A and HR6B, encode highly homologous proteins. Here, we describe the phenotype of cells and mice deficient for the mHR6A gene. Just like mHR6B knockout mouse embryonic fibroblasts, mHR6A-deficient cells appear to have normal DNA damage resistance properties, but mHR6A knockout male and female mice display a small decrease in body weight. The necessity for at least one functional mHR6A (X-chromosomal) or mHR6B (autosomal) allele in all somatic cell types is supported by the fact that neither animals lacking both proteins nor females with only one intact mHR6A allele are viable. In striking contrast to mHR6B knockout males, which show a severe spermatogenic defect, mHR6A knockout males are normally fertile. However, mHR6A knockout females fail to produce offspring despite a normal ovarian histology and ovulation. The absence of mHR6A in oocytes prevents development beyond the embryonic two-cell stage but does not result in an aberrant methylation pattern of histone H3 at this early stage of mouse embryonic development. These observations support redundant but dose-dependent roles for HR6A and HR6B in somatic cell types and germ line cells in mammals.Repair of damaged DNA requires a number of complementary, partially overlapping mechanisms, including mismatch repair, nucleotide and base excision repair, and repair pathways that deal with double-strand breaks. S-phase arrest caused by unrepaired DNA damage is prevented by a mechanism known as postreplication repair, which we prefer to rename replicational damage bypass (RDB). This bypass mechanism allows replication across lesions instead of repairing lesions (20,26).Based on the mutant phenotype, RAD6 is one of the key players in RDB in yeast. rad6 mutants are extremely sensitive to a wide range of DNA-damaging agents and show an increased spontaneous mutation frequency and a loss of DNA damage-induced mutagenesis (41,46). In addition, RAD6 is also involved in a number of other cellular processes in yeast, including the degradation of proteins via the N-end rule, retrotransposition, gene silencing, meiosis, and sporulation (14, 15, 28, 52). RAD6 was identified as a ubiquitin-conjugating enzyme (29), and all of the functions of RAD6 in yeast depend on its ability to transfer ubiquitin molecules to a protein substrate (66). The ubiquitin-conjugating pathway requires the consecutive activity of ubiquitin-activating (E1), -conjugating (E2), and -ligating (E3) enzymes and results in mono-or polyubiquitination of substrate proteins. The ubiquitination serves as a signal for modification, activation, or proteolytic degradation via the 26S proteasome. The identification of 11 ubiquitinconjugating enzymes in yeast and at least 20 genes encoding such proteins in the human genome, as well as the existence of a vast number of E3s, suggests that high substrate specific...