I. NMR study on the interactions of the SeqA protein with hemimethylated and fully methylated DNA
Replication of Escherichia coli chromosomal DNA initiates at a unique site, oriC, and occurs precisely once per cell cycle. Methylation of oriC by Dam methylase, which methylates $N^6$ of adenine in the GATC sequences, enables SeqA to negatively regulate both primary and secondary replication initiation. SeqA delays the primary replication initiation at fully methylated oriC until a cell reaches its critical mass, counteracting the DnaA protein that activates replication initiation. After replication is initiated, SeqA specifically binds the hemimethylated GATC sites, as the daughter strands are transiently unmethylated, and sequesters oriC from Dam methylase and DnaA, thus preventing secondary replication initiation within a single cell cycle. In the absence of SeqA, E. coli cells are viable, but initiation of DNA replication is asynchronous and occurs several times during each cell cycle. In the present study, our object is to examine the dynamics of SeqA with binding of DNAs. The DNAs used in this study contain the hemimethylated and fully methylated GATC sequence. The flexibility of a protein has been suggested as an important factor in a protein-ligand or a protein-nucleic acid interaction. In the crystal structure of the SeqA (51-181 amino acids) with the hemimethylated DNA, it was known that the hemimethylated $^mA:T$ pair of the DNA associates with the L3 loop of SeqA. Although the crystal structure of the SeqA C-terminal segment (51-181 amino acids) has been determined in a complex with hemimethylated DNA, it is meaningful to examine the dynamics of SeqA with DNA in order to understand the precise protein-DNA interaction. To identify the possibility of the progress of this object, we confirmed the $^1H^{15}N$ HSQC spectrum of SeqA alone with well dispersed pattern and carried out the chemical shift perturbation experiment.
II. NMR study of the interaction between WRN and RPA
Werner syndrome (WS) is an autosomal recessive disease that results in premature aging. Mutations in the WS gene (WRN) result in a loss of expression of the WRN protein and predispose WS patients to accelerated aging. As a helicase and a nuclease, WRN is unique among the five human RecQ helicase family members and is capable of multiple functions involved in DNA replication, repair, recombination, and telomere maintenance. A 144-residue fragment of WRN (residues 949-1092) was previously determined to be a multifunctional DNA- and protein-binding domain (DPBD) that interacts with structure-specific DNA and a variety of DNA-processing proteins. There are WRN-protein interactions reported, which result in functional consequences in either WRN or its interactive protein, and each of these interactions requires the DPBD of WRN or a part of the DPBD. One of the WRN-interacting proteins to be recognized is RPA, which is a stable complex of three subunits of 70, 32, and 14 kDa. The activity of WRN helicase is stimulated by human replication protein A. In the present study, WRN and RPA were prepared as soluble form. And the RPA binding surfaces on uniformly $^{15}N$ -labeled WRN were investigated by chemical shift perturbation experiment to find out the details of how interactions between WRN and RPA occur.
Werner syndrome (WS)은 genetic instability로 인해 premature aging이 일어나는 유전질환이다. WS 유전자의 결함으로 WRN (Werner syndrome protein)의 결핍이 생기게 되고, 이 경우에 사춘기에 이를 때까지는 아무런 증상이 나타나지 않다가 20살 정도가 되면 백내장, 탈모, 피부 주름, 골다공증, 동맥경화, 당뇨병 등 온갖 노화의 증상들이 나타나기 시작한다. WRN 단백질은 RecQ helicase 라는 단백질 패밀리에 속한다. 인간 유전체에서 이 패밀리에 속하는 단백질은 다섯 개 발견되었으며, 이들은 DNA 복제, 재조합, 수선 등의 과정에서 중요한 기능을 가지고 있다. DNA의 수선 과정에서 helicase 가 DNA 이중나선을 열어주면 핵산가수분해 효소가 잘못된 부분을 분해하여 제거한다. 그러나 WRN은 다른 helicase 와 달리 한 단백질에 핵산가수분해 활성까지 가지고 있으며 이중가닥 및 단일가닥 단절 부분, 복제 분기점과 교차점, 그리고 심지어는 DNA-RNA 이중가닥에 이르기까지 다양한 형태에서 수선에 관여한다. 2005년에는 WRN의 DNA- and protein-binding domain (DPBD)의 solution structure가 결정되었으며, 이 domain은 다양한 DNA 관련 단백질과 상호작용한다고 알려져 있다. WRN과 상호작용하는 단백질의 하나로써, hRPA (human replication protein A)는 WRN의 helicase의 활성을 높여주며, 이는 WRN과 RPA 사이에 physical interaction이 있음을 의미한다.
이번 연구에서는, WRN에 결합하는 RPA의 결합부위를 결정하기 위하여 NMR 분광법에서 chemical shift perturbation 실험을 이용한 탐구가 이루어졌다. WRN과 RPA 결합에 대한 mapping 결과들을 토대로 실험에서 사용될 domain을 결정하였다. 그리고 $^{15}N-WRN$를 과량 발현, 정제하여 HSQC 를 찍고, RPA의 titration 실험을 통해 WRN과 RPA 사이의 결합을 확인하였다. 그러나 RPA의 titration 에 의해 $^{15}N-WRN$의 피크가 사라지는 결과가 나타나 chemical shift mapping 을 진행할 수 없었다. 그래서 앞으로 WRN과 RPA의 complex 크리스탈 결정을 시도하여, 두 단백질간의 상호작용이 어떻게 일어나는지 알아보고자 한다.
