Maintenance of genome integrity during DNA replication is crucial to the perpetuation of all organisms. In eukaryotes, the bypass of DNA lesions by the replication machinery prevents prolonged stalling of the replication fork, which could otherwise lead to greater damages such as gross chromosomal rearrangements. Bypassing DNA lesions and subsequent repair are accomplished by the activation of DNA damage tolerance pathways such as the template switching (TS) pathway. In yeast, the RAD5 (Radiation-sensitive 5) protein plays a crucial role in initiating the TS pathway by catalyzing the polyubiquitination of PCNA (Proliferation Cell Nuclear Antigen). Likewise, one of the mammalian RAD5-homologs, SHPRH (SNF2, histone linker, PHD, RING, helicase) mediates PCNA polyubiquitination. To date, the study of SHPRH enzymatic functions has been limited to this modification. It is therefore unclear how SHPRH carries out its function in DNA repair. Moreover, how this protein regulates gene transcription at the enzymatic level is also unknown. Given that SHPRH harbors domains found in chromatin remodeling proteins, we investigated its biochemical properties in the presence of nucleosomal substrates. We find that SHPRH binds equally well to double-stranded (ds) DNA and to nucleosome core particles, however, like ISWI and CHD-family remodelers, SHPRH shows a strong preference for nucleosomes presenting extranucleosomal DNA. Moreover, nucleosomes but not dsDNA strongly stimulate the ATPase activity of SHPRH. Intriguingly, unlike typically observed with SNF2-family enzymes, ATPase activity does not translate into conventional nucleosome remodeling, under standard assay conditions. To test whether SHPRH can act as a ubiquitin E3 ligase for nucleosomes, we performed a screen using 26 E2-conjugating enzymes. We uncover that SHPRH is a potent nucleosome E3-ubiquitin-ligase that can function with at least 7 different E2s. Mass spectrometry analyses of products generated in the presence of the UBE2D1-conjugating enzyme reveal that SHPRH can catalyze the formation of polyubiquitin linkages that are either branched or associated with the recruitment of DNA repair factors, as well as linkages involved in proteasomal degradation. We propose that, in addition to polyubiquitinating PCNA, SHPRH promotes DNA repair or transcriptional regulation in part through chromatin ubiquitination. Our study sets a biochemical framework for studying other RAD5- and RAD16-related protein functions through the ubiquitination of nucleosomes.

中文翻译：

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DNA修复蛋白SHPRH是核小体刺激的ATPase和核小体E3泛素连接酶。

在DNA复制过程中维持基因组完整性对于所有生物的永续生存至关重要。在真核生物中，复制机制绕过DNA损伤可防止复制叉长时间停滞，否则可能导致更大的损害，例如总体染色体重排。绕过DNA损伤和随后的修复是通过激活DNA损伤耐受途径（例如模板转换（TS）途径）来完成的。在酵母中，RAD5（辐射敏感5）蛋白通过催化PCNA（增殖细胞核抗原）的多泛素化作用，在启动TS途径中起着至关重要的作用。同样，哺乳动物RAD5-同源物之一SHPRH（SNF2，组蛋白接头，PHD，RING，解旋酶）介导PCNA多聚泛素化。迄今为止，SHPRH酶功能的研究仅限于这种修饰。因此，尚不清楚SHPRH如何在DNA修复中发挥作用。此外，还不清楚该蛋白如何在酶促水平上调节基因转录。鉴于SHPRH包含在染色质重塑蛋白中发现的结构域，我们在存在核小体底物的情况下研究了其生物化学特性。我们发现SHPRH与双链（ds）DNA和核小体核心颗粒均具有良好的结合，但是，像ISWI和CHD家族重塑剂一样，SHPRH对呈现核小体外DNA的核小体表现出强烈的偏好。此外，核小体而非dsDNA强烈刺激SHPRH的ATPase活性。有趣的是，与SNF2家族酶通常观察到的不同，在标准测定条件下，ATPase活性不会转化为常规的核小体重塑。为了测试SHPRH是否可以充当核小体的泛素E3连接酶，我们使用26种E2结合酶进行了筛选。我们发现SHPRH是一种有效的核小体E3-泛素连接酶，可以与至少7种不同的E2共同发挥作用。在UBE2D1共轭酶存在下产生的产物的质谱分析表明，SHPRH可以催化形成分支或与募集DNA修复因子相关的多聚泛素连接，以及与蛋白酶体降解有关的连接。我们提出，除了多泛素化PCNA，SHPRH还部分通过染色质泛素化促进DNA修复或转录调控。