Slowing of the polyomavirus DNA replication fork in response to DDR

一些DNA病毒利用细胞DNA损伤反应(DDR)途径帮助病毒DNA复制(如HSV和HPV)，而另一些病毒可能减弱DDR反应(如腺病毒)，甚至被DDR抑制DNA复制(如EBV和多瘤病毒(PYV))。PYV DNA复制是一种重要的宿主细胞DNA复制的简化模型，它利用单个病毒蛋白(LT)进行来源识别和解旋酶功能，否则招募细胞DNA复制蛋白来复制其病毒基因组。PYV DNA复制所需的主要关键相互作用是LT与细胞单链DNA结合复合体(RPA)和DNA聚合酶α-Primase(Polprim)之间的三个相互作用，这三个相互作用都是启动合成所必需的。我们已经在活细胞和体外确定了DDR阻止病毒DNA复制的条件，并表明这是由ATR介导的，并与这三种蛋白质的磷酸化有关，特别是：Lt，RPA的第二亚单位，和Polprim的第二亚单位。初步研究表明，LT的磷酸化(通过产生模拟磷化突变来评估)不影响LT的大部分功能(DNA结合、六聚体、ATPase、与RPA和Polprim的结合、Polprim对聚合的刺激)，但它显著抑制DNA解旋酶的进展。在不依赖于对LT的这种DDR效应的第二条途径中，引物的合成是不足的。从DDR激活的细胞中纯化的RPA对朴素LT和Polprim的引物合成有严重的抑制作用，这表明在复制分叉处，分别通过解旋酶和引发抑制对领先链和滞后链的合成有协同抑制作用。本建议的两个目的是制备RPA和Polprim配合物在其DDR位点上的仿磷突变，并像我们对仿磷LT所做的那样评估这两个配合物的功能。它们单独的功能，以及结合其他两个复合体的wt和仿磷突变，将阐明复制分叉进程如何以协调的方式抑制DDR背后的详细机制。这对DNA复制压力如何被用于癌症治疗和人类PYV感染的治疗产生了影响。

Some DNA viruses utilize cellular DNA damage response (DDR) pathways to aid in viral DNA replication (e.g. HSV and HPV), while others may attenuate the DDR response (e.g. adenovirus), or even be subject to DNA replication arrest by DDR (e.g. EBV and polyomavirus (PyV)). PyV DNA replication has been an important simplified model of host cell DNA replication that utilizes a single viral protein (LT) for origin recognition and helicase function, and otherwise recruits cellular DNA replication proteins to replicate its viral genomes. The primary critical interactions required for PyV DNA replication are the three interactions between LT and the cellular single-strand DNA binding complex (RPA) and DNA polymerase alpha- primase (Polprim), all three interactions are required for synthesis to be initiated. We have identified conditions both in living cells and in vitro where DDR prevents viral DNA replication, and shown that this is mediated by ATR, and correlates with phosphorylation of each of these three proteins, specifically: LT, the second subunit of RPA, and the second subunit of Polprim. Preliminary studies shown herein demonstrate that the phosphorylation of LT (evaluated by creating phosphomimetic mutations) doesn’t affect most functions of LT (DNA binding, hexamerization, ATPase, binding to RPA and Polprim, stimulation of polymerization by Polprim), but it does dramatically inhibit DNA helicase progression. Synthesis of primers is deficient in a second pathway independent of this DDR effect on LT. Naïve LT and Polprim with RPA purified from DDR-activated cells is severely inhibited for primer synthesis, suggesting that at the replication fork there is a coordinated inhibition of both leading and lagging strand synthesis, through helicase and priming suppression, respectively. The two Aims of this proposal are to prepare phosphomimetic mutations of both the RPA and Polprim complexes at their DDR sites, and evaluate the function of these two complexes as we have done with the phosphomimetic LT. Their functions alone, as well as in conjunction with both wt and phosphomimetic mutations of both of the other complexes will elucidate the detailed mechanisms behind how replication fork progression can be inhibited in a coordinated fashion in response to DDR. This has ramifications on how DNA replication stress can be targeted for cancer treatment and for treatment of human PyV infections.