Oxidative DNA Damage And Its Processing

氧化损伤主要通过碱基切除修复(BER)途径从DNA中移除。BER是通过四个酶步骤进行的，但现在很清楚，其他几种蛋白质通过蛋白质-蛋白质相互作用来调节BER效率。我们和其他人确定了核心BER酶的几种蛋白质相互作用。这些蛋白质相互作用是物理和功能的，共同支持“传递接力棒”模型，在该模型中，BER在不同步骤中发生，由单个蛋白质相互作用支持，这些蛋白质相互作用是修复复合体的组成部分，可能位于DNA损伤处。我们正在研究OGG1的其他蛋白质相互作用，以了解氧化损伤的修复在体内是如何调控的。我们发现OGG1也与重组蛋白RAD52相互作用，这表明这两个修复途径之间可能存在相互作用。我们发现这两种蛋白质之间存在功能上的相互作用，其中RAD52刺激OGG1的催化活性，而OGG1抑制RAD52催化的DNA链的退火和侵袭。此外，在暴露于氧化应激的细胞中，OGG1和RAD52之间的物理相互作用增加，表明这种相互作用在细胞对氧化DNA损伤的反应中是重要的。 最近，我们发现OGG1在中风后的康复过程中也起着重要作用。使用OGG1基因敲除(KO)小鼠模型，我们评估了OGG1在永久性大脑中动脉闭塞后的重要性，这是一种中风模型。我们观察到OGG1KO小鼠的皮质梗塞面积和行为缺陷比WT小鼠更大。此外，在体外，从OGG1KO小鼠分离的皮质神经元更容易受到氧化损伤。我们的结果表明，修复氧化产生的DNA损伤的能力与神经元对代谢和氧化应激的脆弱性有关，此外还与中风后的功能结果有关。 XRCC1是单链断裂修复所必需的支架蛋白。在人类中，XRCC1的缺陷会导致染色体不稳定，并增加对DNA损伤剂的敏感性。最近，我们对XRCC1的七个不同的定点突变的催化活性和蛋白质相互作用能力进行了评估，以探讨它们的功能意义。我们发现有两个影响了蛋白质的完整性，一个干扰了XRCC1与Pol Beta的相互作用，四个没有表型。通过这样的分析，我们希望确定各种基于疾病和人群的XRCC1变异如何影响与XRCC1相关的疾病表型。

Oxidative lesions are removed from DNA primarily via the base excision repair (BER) pathway. BER is carried out through four enzymatic steps, but it is now clear that several other proteins modulate BER efficiency through protein-protein interactions. We and others identified several protein interactions for the core BER enzymes. These protein interactions are physical and functional and together support the "passing of baton" model, in which BER takes place in different steps supported by individual protein interactions that are components of a repair complex, possibly situated at the DNA lesion. We are studying other protein interactions of OGG1 in order to understand how repair of oxidative lesions is regulated in vivo. We find that OGG1 also interacts with the recombination protein RAD52, suggesting a possible interplay between these two repair pathways. We find a reciprocal functional interaction between these two proteins, in which RAD52 stimulates OGG1 catalytic activity and OGG1 inhibits RAD52-catalysed DNA strand annealing and invasion. Moreover, the physical interaction between OGG1 and RAD52 increases in cells exposed to oxidative stress, indicating that this interaction is important in the cellular response to oxidative DNA damage. More recently we have shown that OGG1 also plays a significant role in the recovery following a stroke. Using the OGG1 knockout (KO) mouse model, we evaluated the importance of OGG1 after permanent middle cerebral artery occlusion, a stroke model. We observed larger cortical infarcts areas and behavior deficits in the OGG1 KO mice than in WT mice. Additionally, in vitro, the cortical neurons isolated from OGG1 KO mice were more vulnerable to oxidative insults. Our results suggest that the ability to repair oxidatively generated DNA damage has implications for the vulnerability of neurons to metabolic and oxidative stress, and additionally on the functional outcomes after stroke. XRCC1 is an essential scaffold protein for single strand break repair. In humans, deficiencies in XRCC1 lead to chromosome instability and increased sensitivity to DNA damaging agents. Recently we evaluated the catalytic activities and protein interaction capabilities of seven different site directed mutants of XRCC1 to investigate their functional significance. We found two that compromised the integrity of the protein, one interfered with XRCC1s interaction with Pol Beta and four had no phenotype. Through such analysis we hope to define how various disease and population based XRCC1 variants might contribute to disease phenotypes associated with XRCC1.