研究N-WASP在基因组复制中的作用及机制是重要的科学问题。我们前期研究发现N-WASP敲除小鼠显著抑制皮肤肿瘤生成。N-WASP缺失的细胞展现出基因组复制缺陷和细胞衰老。基因组复制缺陷会导致细胞衰老而抑制早期细胞癌变。Co-IP发现N-WASP能结合PCNA，PCNA是DNA聚合酶的重要辅助因子，提示N-WASP可能通过调控PCNA而调控基因组复制从而在肿瘤发生中发挥重要作用。我们推测N-WASP可能通过以下四个途径调控PCNA：调节PCNA蛋白稳定性或翻译后修饰；影响PCNA结合蛋白的选择；通过表观遗传H3K56me1调控PCNA结合染色质及复制中定位。我们将在N-WASP缺失细胞中检测PCNA表达，翻译后修饰及其在基因组复制中的定位，并利用二级质谱定量研究PCNA结合蛋白谱的变化，以阐明N-WASP调控PCNA的分子机制。研究结果对于理解基因组复制调控网络和肿瘤发生具有重要意义。

Revealing the molecular mechanism how N-WASP regulates genome replication integrity is an important scientific question. Our preliminary data showed that N-WASP knockout mice are highly resistant to skin tumor induction. Primary cultured N-WASP knockout keratinocytes exhibited a defect in genome replication and cellular senescence. Genome replication defect is one of the important inducers of cellular senescence, which is a major inhibitory mechanism preventing early tumor transformation. By Co-IP, N-WASP was found to bind PCNA, an essential co-factor for DNA polymerases involved in regulating genome replication integrity. Together, these data suggest N-WASP may exert its important role in skin tumor formation by regulating PCNA to maintain replication integrity. We speculate N-WASP could modulate PCNA via 4 mechanisms: regulating PCNA stability and post-translational modifications; influencing PCNA's binding protein; facilitating PCNA binding to chromatin through H3K56me1. In this study, we will use N-WASP knockout primary keratinocytes to check PCNA protein level, post-translational modification including but not limited to ubiquitination and sumoylation, sub-cellular localization during genome replication, and quantify its binding protein changes using LC-MS/MS. We expect to elucidate the molecular mechanism how N-WASP regulates PCNA to maintain replication integrity. Results generated here will further extend our understanding of the replication regulatory network and uncover a novel mechanism of tumorigenesis.