Satyanarayana K, Rao M R
Department of Biochemistry, Indian Institute of Science, Bangalore.
Biochem J. 1989 Aug 1;261(3):775-86. doi: 10.1042/bj2610775.
Poly(ADP-ribosyl)ation of nuclear proteins was several-fold higher in the pachytene spermatocytes than in the premeiotic germ cells of the rat. Among the histones of the pachytene nucleus, histone subtypes H2A, H1 and H3 were poly(ADP-ribosyl)ated. Based on the immunoaffinity fractionation procedure of Malik, Miwa, Sugimara & Smulson [(1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2554-2558] we have fractionated DNAase-II-solubilized chromatin into poly(ADP-ribosyl)ated chromatin (PAC) and non-poly(ADP-ribosyl)ated chromatin (non-PAC) domains on an anti-[poly(ADP-ribose)] IgG affinity matrix. Approx. 2.5% of the pachytene chromatin represented the PAC domains. A significant amount of [alpha-32P]dATP-labelled pachytene chromatin (labelled in vitro) was bound to the affinity matrix. The DNA of pachytene PAC domains had internal strand breaks, significant length of gaps and ligatable ends, namely 5'-phosphoryl and 3'-hydroxyl termini. On the other hand, the PAC domains from 18 h regenerating liver had very few gaps, if any. The presence of gaps in the pachytene PAC DNA was also evident from thermal denaturation studies. Although many of the polypeptides were common to the PAC domains of both pachytene and regenerating liver, the DNA sequences associated with these domains were quite different. A 20 kDa protein and the testis-specific histone H1t were selectively enriched in the pachytene PAC domains. The pachytene PAC domains also contained approx. 10% of the messenger coding sequences present in the DNAase-II-solubilized chromatin. The pachytene PAC domains, therefore, may represent highly enriched DNA-repair domains of the pachytene nucleus.
大鼠粗线期精母细胞中核蛋白的聚（ADP - 核糖基）化水平比减数分裂前生殖细胞中的高几倍。在粗线期细胞核的组蛋白中，组蛋白亚型H2A、H1和H3发生了聚（ADP - 核糖基）化。基于Malik、Miwa、Sugimara和Smulson [（1983年）美国国家科学院院刊80，2554 - 2558]的免疫亲和分级分离程序，我们在抗[聚（ADP - 核糖）]IgG亲和基质上，将DNA酶II可溶解的染色质分离为聚（ADP - 核糖基）化染色质（PAC）和非聚（ADP - 核糖基）化染色质（非PAC）结构域。大约2.5%的粗线期染色质代表PAC结构域。大量[α - 32P]dATP标记的粗线期染色质（体外标记）与亲和基质结合。粗线期PAC结构域的DNA有内部链断裂、显著长度的缺口和可连接末端，即5'-磷酸和3'-羟基末端。另一方面，18小时再生肝的PAC结构域几乎没有缺口（如果有的话）。粗线期PAC DNA中缺口的存在从热变性研究中也很明显。虽然粗线期和再生肝的PAC结构域有许多共同的多肽，但与这些结构域相关的DNA序列却大不相同。一种20 kDa的蛋白质和睾丸特异性组蛋白H1t在粗线期PAC结构域中选择性富集。粗线期PAC结构域还包含DNA酶II可溶解染色质中约10%的信使编码序列。因此，粗线期PAC结构域可能代表粗线期细胞核中高度富集的DNA修复结构域。
