MECHANISMS OF THE HSV-1 UL42 PROTEIN

在高度多样化的生物体中，DNA复制的需求和机制有着惊人的一致性。单纯疱疹病毒1型（HSV-1）是真核生物DNA复制的一个极好的模型系统，因为该病毒编码这一基本过程所需的大多数蛋白质，并且比高级真核生物更容易进行遗传操作。DNA聚合酶是DNA复制过程的核心。复制性DNA聚合酶的一般要求是它们复制模板基因组的速度和合理的保真度。它们达到基因组复制所需的必要速率的一个主要手段是使用辅助蛋白来增加它们的加工能力。HSV-1 DNA聚合酶（pol）与辅助因子UL42形成稳定的特异性复合物。像其他pol辅助蛋白一样，UL42增加了其同源pol的处理能力，但在几个重要方面有所不同。它不需要夹子装载蛋白质，这使它与环状滑动夹子（如PCNA和大肠杆菌pol III β）区别开来。此外，它与DNA结合的内在能力在所有其他已知的加工因子中是独一无二的，包括那些不需要箝位装载物的因子，如硫氧还蛋白，T7噬菌体pol的加工因子。后一种能力也为已知的加工能力机制提出了一个明显的悖论，即UL42也可以作为伸长率的刹车。提出的研究的主要长期目标是阐明UL42增加pol处理能力的机制，以及该机制对pol的其他特性的影响，包括DNA复制保真度所需的参数。生物化学、生物物理和遗传方法的结合将用于解决四个具体目标：1)利用瞬态动力学分析和直接结合研究确定突变UL42蛋白减少DNA结合对延伸率和pol加工率的影响；2)通过动力学分析，确定处理能力和校对能力对影响体外保真度的单个参数的影响，包括核苷酸选择、不匹配末端延伸失败和不匹配引物末端切除；3)确定保真度参数变化（由处理性变化引起）对体内起源（ori）依赖性DNA复制过程中发生的突变频率和类型的生物学影响；4)为了确定与UL42相互作用的同源结合蛋白UL9促进pol/UL42复合物在阻断的合成引物/模板上的组装和/或加工能力。所有人类疱疹病毒都能编码HSV-1 pol和UL42的功能类似物，包括卡波西肉瘤相关病毒（HHV-8）、爱泼斯坦巴尔病毒和人类巨细胞病毒，所有这些病毒都是重要的人类病原体，特别是对癌症和免疫抑制患者。了解UL42的作用机制是很重要的，因为pol/UL42复合物的破坏已被提出用于抗病毒药物的开发。

There is amazing conservation in the requirements for and mechanism of DNA replication among highly diverse organisms. Herpes simplex virus type 1 (HSV-1) is an excellent model system for eukaryotic DNA replication since the virus encodes most of the proteins required for this essential process and can be manipulated genetically with greater ease than higher eukaryotes. DNA polymerases are central to the process of DNA replication. A general requirement of replicative DNA polymerases is that they copy the template genome with rapidity and reasonable fidelity. A major means by which they achieve the necessary rate required for genome duplication is the use of accessory proteins to increase their processivity. The HSV-1 DNA polymerase (pol) forms a stable and specific complex with an accessory factor, UL42. Like other pol accessory proteins, UL42 increases the processivity of its cognate pol, but differs in several important ways. Its lack of requirement for clamp loading proteins distinguishes it from the toroid sliding clamps, such as PCNA and E. coli pol III beta. Furthermore its intrinsic ability to bind to DNA is unique among all other known processivity factors, including those which don't require clamp loaders, such as thioredoxin, the processivity factor for T7 bacteriophage pol. The latter ability also presents an apparent paradox for known mechanisms of processivity, in that UL42 could also serve as a brake to elongation. The major long-term goal of the proposed studies is to elucidate the mechanism by which UL42 increases pol processivity, and the resulting impact this mechanism has on other properties of the pol, including parameters required for fidelity of DNA replication. A combination of biochemical, biophysical, and genetic approaches will be used to address four specific aims: 1) To determine the effect of reduced DNA binding by mutant UL42 proteins on rates of elongation and pol processivity using transient kinetic analysis and direct binding studies; 2) To determine the effect of processivity and proof-reading capability on the individual parameters which affect fidelity in vitro, including nucleotide selection, failure to extend mismatched termini, and excision of mismatched primer termini, using kinetic analysis to dissect these processes; 3) To determine the biological impact of changes in fidelity parameters (caused by changes in processivity) on the frequency and types of mutations which occur during origin (ori)- dependent DNA replication in vivo; 4) To determine the ability of the ori-binding protein, UL9, which interacts with UL42, to facilitate the assembly and/or processivity of pol/UL42 complexes on blocked synthetic primer/templates. Functional analogs of HSV-1 pol and UL42 are encoded by all human herpesviruses, including Kaposi sarcoma-associated virus (HHV-8), Epstein Barr virus, and human cytomegalovirus, all of which are significant human pathogens, particularly for cancer and immuno- suppressed patients. It is important to understand an the mechanism of UL42 action since disruption of the pol/UL42 complex has been proposed for development of antivirals.