Presence of nuclear factors bound to both cAMP-responsive element and AP1 factor binding site in the porcine anterior pituitary.

Factors binding to consensus sequences of the cAMP-responsive element (CRE) and the AP1 factor binding site (AP1) were investigated using porcine anterior pituitary nuclear extracts. Each element showed specific gel mobility shifts. By reciprocal competition for the AP1 and CRE binding, CRE prevented AP1 binding completely. On the other hand, AP1 decreased the CRE binding considerably to 20%, suggesting that approximately 80% of the total CRE binding is due to factors which bind to a common site shared by both CRE and AP1, whereas proteins binding to AP1 alone are absent. Relative binding affinities of AP1 against CRE estimated from the reciprocal competition data were 0.17 for CRE binding and 0.56 for AP1 binding. UV cross-linking experiments showed that CRE and AP1 gave different patterns consisting of different molecular size. Inconsistency of the relative binding affinities and the multiple molecular size of binding factors, cannot be explained simply by the presence of two types of binding factor, common CRE/AP1-binding and specific CRE-binding factors. A more likely explanation is that the CRE/AP1-binding factors alter the dimer form by changing each respective partner to bind CRE and/or AP1.     

胡萝卜AP2／ERF-B1亚族两个转录因子基因的克隆及其非生物胁迫响应分析

AP2／ERF是植物中普遍存在的一类重要转录因子,参与植物整个生命周期的生长发育和逆境信号转导。本研究以胡萝卜（Daucus carota）‘黑田五寸’为试验材料,基于其转录组和基因组数据,检索和拼接获得胡萝卜AP2／ERF家族2个转录因子基因序列g39811和g47170。采用RT-PCR方法,分别从‘黑田五寸’中克隆DcERF-B1-1（g39811）和DcERF-B1—2（g4717D）转录因子基因。序列分析显示,胡萝卜DcERF-B1-1和DcERF-B1-2转录因子基因分别含有630个和594个开放阅读框,分别编码209和197个氨基酸;均含有相对保守的AP2结合域,具有典型的植物AP2／ERF类转录因子特征。从氨基酸组成成分、理化性质、亲水性／疏水性和三级结构上分析显示,胡萝卜DcERF—B1-1和DcERF-B1-2转录因子亲水性大于疏水性,其氨基酸序列可能属于亲水性蛋白。空间结构分析显示,它们都具有1个α螺旋和3个β折叠。进化树分析显示,二者均属于AP2／ERF家族转录因子中ERF亚族的B1组。实时定量荧光PCR显示,在低温、干旱、盐胁迫的条件下,DcERF-B1-2转录因子比DcERF-B1-1转录因子对逆境的响应更大;在高温的条件下,DcERF-B1-1转录因子比DcERF-B1-2转录因子对逆境的响应更大。     

The USP1-UAF1 complex interacts with RAD51AP1 to promote homologous recombination repair

USP1 deubiquitinating enzyme and its stoichiometric binding partner UAF1 play an essential role in promoting DNA homologous recombination (HR) repair in response to various types of DNA damaging agents. Deubiquitination of FANCD2 may be attributed to the key role of USP1-UAF1 complex in regulating HR repair, however whether USP1-UAF1 promotes HR repair independently of FANCD2 deubiquitination is not known. Here we show evidence that the USP1-UAF1 complex has a FANCD2-independent function in promoting HR repair. Proteomic search of UAF1-interacting proteins revealed that UAF1 associates with RAD51AP1, a RAD51-interacting protein implicated in HR repair. We show that UAF1 mediates the interaction between USP1 and RAD51AP1, and that depletion of USP1 or UAF1 led to a decreased stability of RAD51AP1. Protein interaction mapping analysis identified some key residues within RAD51AP1 required for interacting with the USP1-UAF1 complex. Cells expressing the UAF1 interaction-deficient mutant of RAD51AP1 show increased chromosomal aberrations in response to Mitomycin C treatment. Moreover, similar to the RAD51AP1 depleted cells, the cells expressing UAF1-interaction deficient RAD51AP1 display persistent RAD51 foci following DNA damage exposure, indicating that these factors regulate a later step during the HR repair. These data altogether suggest that the USP1-UAF1 complex promotes HR repair via multiple mechanisms: through FANCD2 deubiquitination, as well as by interacting with RAD51AP1.