Yao Nina, Coryell Lee, Zhang Dan, Georgescu Roxana E, Finkelstein Jeff, Coman Maria M, Hingorani Manju M, O'Donnell Mike
The Rockefeller University, New York, New York 1002, USA.
J Biol Chem. 2003 Dec 12;278(50):50744-53. doi: 10.1074/jbc.M309206200. Epub 2003 Oct 6.
Replication factor C (RFC) is a heteropentameric AAA+ protein clamp loader of the proliferating cell nuclear antigen (PCNA) processivity factor. The prokaryotic homologue, gamma complex, is also a heteropentamer, and structural studies show the subunits are arranged in a circle. In this report, Saccharomyces cerevisiae RFC protomers are examined for their interaction with each other and PCNA. The data lead to a model of subunit order around the circle. A characteristic of AAA+ oligomers is the use of bipartite ATP sites in which one subunit supplies a catalytic arginine residue for hydrolysis of ATP bound to the neighboring subunit. We find that the RFC(3/4) complex is a DNA-dependent ATPase, and we use this activity to determine that RFC3 supplies a catalytic arginine to the ATP site of RFC4. This information, combined with the subunit arrangement, defines the composition of the remaining ATP sites. Furthermore, the RFC(2/3) and RFC(3/4) subassemblies bind stably to PCNA, yet neither RFC2 nor RFC4 bind tightly to PCNA, indicating that RFC3 forms a strong contact point to PCNA. The RFC1 subunit also binds PCNA tightly, and we identify two hydrophobic residues in RFC1 that are important for this interaction. Therefore, at least two subunits in RFC make strong contacts with PCNA, unlike the Escherichia coli gamma complex in which only one subunit makes strong contact with the beta clamp. Multiple strong contact points to PCNA may reflect the extra demands of loading the PCNA trimeric ring onto DNA compared with the dimeric beta ring.
复制因子C（RFC）是增殖细胞核抗原（PCNA）持续性因子的一种异源五聚体AAA +蛋白钳加载器。其原核同源物γ复合体也是一种异源五聚体，结构研究表明这些亚基呈环状排列。在本报告中，对酿酒酵母RFC原体之间及其与PCNA的相互作用进行了研究。这些数据得出了一个关于环状亚基顺序的模型。AAA +寡聚体的一个特点是使用二分ATP位点，其中一个亚基提供一个催化精氨酸残基用于水解与相邻亚基结合的ATP。我们发现RFC(3/4)复合体是一种依赖DNA的ATP酶，并且我们利用这种活性确定RFC3为RFC4的ATP位点提供一个催化精氨酸。这些信息与亚基排列相结合，确定了其余ATP位点的组成。此外，RFC(2/3)和RFC(3/4)亚组装体与PCNA稳定结合，但RFC2和RFC4都不与PCNA紧密结合，这表明RFC3与PCNA形成了一个强接触点。RFC1亚基也与PCNA紧密结合，并且我们在RFC1中鉴定出两个对这种相互作用很重要的疏水残基。因此，与大肠杆菌γ复合体不同，RFC中至少有两个亚基与PCNA形成强接触，在大肠杆菌γ复合体中只有一个亚基与β钳形成强接触。与二聚体β环相比，与PCNA的多个强接触点可能反映了将PCNA三聚体环加载到DNA上的额外需求。
