9月9日出版的Cell雜志封面是一雙錯配的襪子,，喻意是關(guān)于DNA錯配和修復(fù)的,。文章的通訊作者為華人科學(xué)家——李國民（音譯）,，在武漢大學(xué)生物系獲得理學(xué)學(xué)士學(xué)位,，在美國Wayne State University (韋恩州立大學(xué))取得理學(xué)博士學(xué)位,，目前是美國肯塔基大學(xué)醫(yī)學(xué)中心（University of Kentucky Medical Center）的副教授,。
李國民教授多年來從事DNA復(fù)制錯配與修復(fù)的分子機(jī)制研究,。DNA除了外界環(huán)境影響會造成損傷外，在其本身的復(fù)制過程中也會出現(xiàn)錯配,。盡管這種出錯的幾率十分微小,，但是生命這臺精密的儀器不允許任何差錯。因為就算是一個堿基的錯配也可能造成嚴(yán)重的疾病,。如果錯配修復(fù)基因存在缺陷的話就可能造成癌癥,，比如：遺傳性非多發(fā)性息肉結(jié)腸癌（hereditary nonpolyposis colorectal cancer）。
對于復(fù)制過程中出現(xiàn)的錯誤,，比如堿基錯配,、少量插入、缺失,，機(jī)體內(nèi)本身就存在有校正機(jī)制,。這種校正機(jī)制通常可以由DNA聚合酶3’→5’的校對功能立即進(jìn)行糾正,。如果DNA聚合酶沒有發(fā)現(xiàn)錯配,，就需要錯配修復(fù)（mismatch repair,
MMR）系統(tǒng)來完成。
在大腸桿菌中的錯配修復(fù)系統(tǒng)已經(jīng)研究得十分透徹了,。這個系統(tǒng)主要包括酶錯配矯正酶（mismatch correction enzyme）,、DNA聚合酶Ⅲ和DNA連接酶等11種蛋白參與?？梢苑譃椴襟E：啟始,、切除和修復(fù)。啟始：根據(jù)序列上GATC中腺嘌呤（A）的甲基化程度不同,，酶錯配矯正酶中的MutS部分識別錯配區(qū)域,，而MutH和MutL識別沒有甲基化的GATC序列。之后,，將新合成的單鏈上切出一個缺口（nick）,。切除：由核酸外切酶將距離GATC到錯配區(qū)域間的DNA鏈切除。為了防止剩下的暴露的單鏈遭到降解,，需要有單鏈結(jié)合蛋白（Ssb）將其保護(hù)起來,。修復(fù)：由DNA聚合酶Ⅲ全酶進(jìn)行合成，連接酶對缺口進(jìn)行連接,。
錯配修復(fù)系統(tǒng)是一種高度保守的途經(jīng),。理論上,，它在真核系統(tǒng)中的修復(fù)過程應(yīng)該與原核系統(tǒng)中的相類似。原核系統(tǒng)是依靠甲基化程度來判斷哪條是新合成鏈,，而在真核系統(tǒng)中是如何識別錯配單鏈的機(jī)制尚未明確,。但是，人們在體外可以利用含有MutSα或者M(jìn)utSβ,、MutLα,、RPA、PCNA,、EXO1,、HMGB1、RFC和DNA聚合酶δ的系統(tǒng)將含有缺口的雙鏈DNA進(jìn)行修復(fù),。
李國民教授這篇文章Reconstitution of 5′-Directed Human Mismatch Repair in a Purified System，文章利用純化后的人源蛋白在體外對5’方向存在缺口的錯配DNA進(jìn)行了修復(fù),。這一系統(tǒng)包括了MutSα或者M(jìn)utSβ,、MutLα、RPA,、PCNA,、EXO1、HMGB1,、RFC和DNA聚合酶δ蛋白,。在這個系統(tǒng)中，MutSβ對于堿基錯配的修復(fù)作用十分有限,，它對于插入/缺失錯配的修復(fù)率比MutSα高,；MutSα對于兩種類型的錯配修復(fù)率是一樣的。MutLα可以減低EXO1的酶切活性,，并且在錯配區(qū)域被切除后MutLα可以終止EXO1的催化切除效果,。如果缺乏MutLα蛋白EXO1就會無法停止作用而切除過多的DNA。
RPA和HMGB1在MutSα激活的EXO1催化切除的錯配修復(fù)啟始,、切除過程中起到相似但是互補(bǔ)的作用,，但是RPA在協(xié)助MutLα調(diào)節(jié)切除過錯配區(qū)域后的終止過程中起到完全不同的作用。一個堿基有效的錯配修復(fù)需要MutSα-MutLα多分子復(fù)合物的參與,。這一研究描述了一種由錯配引起的DNA切除的啟始和終止模式,。
在體外重新構(gòu)建的錯配修復(fù)系統(tǒng)能執(zhí)行的只是體內(nèi)錯配修復(fù)系統(tǒng)的一部分功能。它必須以出現(xiàn)缺口的雙鏈DNA為底物進(jìn)行反應(yīng),，而在體內(nèi)這種缺口產(chǎn)生的機(jī)制仍不清楚,，特別是先導(dǎo)鏈（leading strand）缺口的產(chǎn)生。在這項研究中將真核系統(tǒng)錯配修復(fù)系統(tǒng)中MutLα的作用機(jī)理進(jìn)行了進(jìn)一步的闡明,，是MutLα使EXO1失去酶切的活性,，從而使切除反應(yīng)終止,。這對于錯配修復(fù)系統(tǒng)是否能正常工作是一個關(guān)鍵的步驟。
李國民教授聯(lián)系方式：
Graduate Center for Toxicology and Markey Cancer Center, University of Kentucky Medical Center, Lexington, Kentucky 40536
Phone: (859) 257-7053
E-mail: [email protected]
研究興趣
Research Interest: Molecular mechanism of DNA mismatch repair.
DNA mismatch repair maintains genomic stability by correcting mismatches generated during normal DNA metabolism and by mediating DNA damage-induced apoptosis. Defects in mismatch repair genes are the genetic basis of certain types of human cancer, including hereditary nonpolyposis colorectal cancer. To fully understand the molecular mechanisms of the mismatch repair system in human cells, our work is focused on three areas: 1) enzymology of mismatch repair; 2) identification and characterization of proteins involved in DNA damage-induced, mismatch repair-dependent apoptosis; and 3) genetic alterations of mismatch repair genes in human cancer and other diseases.
小常識：DNA錯配修復(fù)的機(jī)制
Mismatch repair
To repair mismatched bases, the system has to know which base is the correct one. In E. coli, this is achieved by a special methylase called the "Dam methylase", which can methylate all adenines that occur within (5')GATC sequences. Immediately after DNA replication, the template strand has been methylated, but the newly synthesized strand is not methylated yet. Thus, the template strand and the new strand can be distinguished.
. Mismatch repair.
The repairing process begins with the protein MutS which binds to mismatched base pairs. Then, MutL is recruited to the complex and activates MutH which binds to GATC sequences. Activation of MutH cleaves the unmethylated strand at the GATC site. Subsequently, the segment from the cleavage site to the mismatch is removed by exonuclease (with assistance from helicase II and SSB proteins). If the cleavage occurs on the 3' side of the mismatch, this step is carried out by exonuclease I (which degrades a single strand only in the 3' to 5' direction). If the cleavage occurs on the 5' side of the mismatch, exonuclease VII or RecJ is used to degrade the single stranded DNA. The gap is filled by DNA polymerase III and DNA ligase.
The distance between the GATC site and the mismatch could be as long as 1,000 base pairs. Therefore, mismatch repair is very expensive and inefficient.
Mismatch repair in eukaryotes may be similar to that in E. coli. Homologs of MutS and MutL have been identified in yeast, mammals, and other eukaryotes. MSH1 to MSH5 are homologous to MutS; MLH1, PMS1 and PMS2 are homologous to MutL. Mutations of MSH2, PMS1 and PMS2 are related to colon cancer.
In eukaryotes, the mechanism to distinguish the template strand from the new strand is still unclear.
