DNA合成的忠实性机制

DNA的忠实性合成对于基因组稳定和物种延续至关重要,否则可能会产生严重的后果。DNA合成具有极高的忠实性,这主要基于3个步骤:(1)基于氢键、碱基对构象或其他因素的核苷酸选择;(2)基于3′→5′外切酶活性的校对,方式有顺式校对和反式校对,可以去除错误掺入的核苷酸;(3)基于错配修复、切除修复、同源重组修复和跨损伤DNA合成的修复过程,可以纠正逃过校对的错误核苷酸。由于DNA聚合酶不仅可以作为抗病毒或抗癌药物的靶标,而且其忠实性还与抗药性或药物副作用有关,所以深入研究DNA合成的忠实性具有非常重要的意义。文章主要论述了DNA合成的忠实性机制,并对DNA聚合酶的应用前景做了展望。     

Mismatch-induced conformational distortions in polymerase beta support an induced-fit mechanism for fidelity

Molecular dynamics simulations of DNA polymerase (pol) beta complexed with different incorrect incoming nucleotides (G x G, G x T, and T x T template base x incoming nucleotide combinations) at the template-primer terminus are analyzed to delineate structure-function relationships for aberrant base pairs in a polymerase active site. Comparisons, made to pol beta structure and motions in the presence of a correct base pair, are designed to gain atomically detailed insights into the process of nucleotide selection and discrimination. In the presence of an incorrect incoming nucleotide, alpha-helix N of the thumb subdomain believed to be required for pol beta's catalytic cycling moves toward the open conformation rather than the closed conformation as observed for the correct base pair (G x C) before the chemical reaction. Correspondingly, active-site residues in the microenvironment of the incoming base are in intermediate conformations for non-Watson-Crick pairs. The incorrect incoming nucleotide and the corresponding template residue assume distorted conformations and do not form Watson-Crick bonds. Furthermore, the coordination number and the arrangement of ligands observed around the catalytic and nucleotide binding magnesium ions are mismatch specific. Significantly, the crucial nucleotidyl transferase reaction distance (P(alpha)-O3') for the mismatches between the incoming nucleotide and the primer terminus is not ideally compatible with the chemical reaction of primer extension that follows these conformational changes. Moreover, the extent of active-site distortion can be related to experimentally determined rates of nucleotide misincorporation and to the overall energy barrier associated with polymerase activity. Together, our studies provide structure-function insights into the DNA polymerase-induced constraints (i.e., alpha-helix N conformation, DNA base pair bonding, conformation of protein residues in the vicinity of dNTP, and magnesium ions coordination) during nucleotide discrimination and pol beta-nucleotide interactions specific to each mispair and how they may regulate fidelity. They also lend further support to our recent hypothesis that additional conformational energy barriers are involved following nucleotide binding but prior to the chemical reaction.     

Non-natural nucleotides as probes for the mechanism and fidelity of DNA polymerases

DNA is a remarkable macromolecule that functions primarily as the carrier of the genetic information of organisms ranging from viruses to bacteria to eukaryotes. The ability of DNA polymerases to efficiently and accurately replicate genetic material represents one of the most fundamental yet complex biological processes found in nature. The central dogma of DNA polymerization is that the efficiency and fidelity of this biological process is dependent upon proper hydrogen-bonding interactions between an incoming nucleotide and its templating partner. However, the foundation of this dogma has been recently challenged by the demonstration that DNA polymerases can effectively and, in some cases, selectively incorporate non-natural nucleotides lacking classic hydrogen-bonding capabilities into DNA. In this review, we describe the results of several laboratories that have employed a variety of non-natural nucleotide analogs to decipher the molecular mechanism of DNA polymerization. The use of various non-natural nucleotides has lead to the development of several different models that can explain how efficient DNA synthesis can occur in the absence of hydrogen-bonding interactions. These models include the influence of steric fit and shape complementarity, hydrophobicity and solvation energies, base-stacking capabilities, and negative selection as alternatives to rules invoking simple recognition of hydrogen-bonding patterns. Discussions are also provided regarding how the kinetics of primer extension and exonuclease proofreading activities associated with high-fidelity DNA polymerases are influenced by the absence of hydrogen-bonding functional groups exhibited by non-natural nucleotides.     

Structural insights into DNA polymerase beta deterrents for misincorporation support an induced-fit mechanism for fidelity

DNA polymerases generally select the correct nucleotide from a pool of structurally similar molecules to preserve Watson-Crick base-pairing rules. We report the structure of DNA polymerase beta with DNA mismatches situated in the polymerase active site. This was achieved by using nicked product DNA that traps the mispair (template-primer, A-C or T-C) in the nascent base pair binding pocket. The structure of each mispair complex indicates that the bases do not form hydrogen bonds with one another, but form a staggered arrangement where the bases of the mispair partially overlap. This prevents closure/opening of the N subdomain that is believed to be required for catalytic cycling. The partially open conformation of the N subdomain results in distinct hydrogen bonding networks that are unique for each mispair. These structures define diverse molecular aspects of misinsertion that are consistent with the induced-fit model for substrate specificity.     

RNA病毒基因组和转录复制多样性的分子基础

自然界中RNA病毒的种类和数量比DNA病毒多得多,根据基因组类型,RNA病毒可分为多种类型,许多研究者认为,存在于古细菌Myxobacteria中,仅仅有一个逆转录酶基因的反转子（Retron)可能是所有病毒的祖先,进化的模式如下,反转子→反座子→反转录转座子→反转录病毒→副反转录病毒→DNA病毒,RNA病毒转录。／复制在很多特征上与DNA病毒迥然不同,依赖于RNA的RNA聚合酶是RNA病转录／复制的主要催化剂,RNA病毒基因组转录和复制都从3'端poly(A)或类tRNA结构或其他结构起始,内部终止是转录,通读到5'末端终止是复制,RNA病毒的模板有正链病毒（RNA模板,负链病毒RNA模板和全长正负链反基因组RNA模板,RNA模板的选择调控机制非常复杂,目前知之甚少,选择模板,RNA聚合酶与转录因子结合形成复制体是两种主要的调控方法,另外,5'UTR和3'UTR也可以调控RNA病毒的转录。     

An induced-fit kinetic mechanism for DNA replication fidelity: direct measurement by single-turnover kinetics.

An exonuclease-deficient mutant of T7 DNA polymerase was constructed and utilized in a series of kinetic studies on misincorporation and next correct dNTP incorporation. By using a synthetic oligonucleotide template-primer system for which the kinetic pathway for correct incorporation has been solved [Patel, S.S., Wong, I., & Johnson, K. A. (1991) Biochemistry (first of three papers in this issue)], the kinetic parameters for the incorporation of the incorrect triphosphates dATP, dCTP, and dGTP were determined, giving, respectively, kcat/Km values of 91, 23, and 4.3 M-1 s-1 and a discrimination in the polymerization step of 10(5)-10(6). The rates of misincorporation in all cases were linearly dependent on substrate concentration up to 4 mM, beyond which severe inhibition was observed. Competition of correct incorporation versus dCTP revealed an estimated Ki of approximately 6-8 mM, suggesting a corresponding kcat of 0.14s-1. Moderate elemental effects of 19-, 17-, and 34-fold reduction in rates were measured by substituting the alpha-thiotriphosphate analogues for dATP, dCTP, and dGTP, respectively, indicating that the chemistry step is partially rate-limiting. The absence of a burst of incorporation during the first turnover places the rate-limiting step at a triphosphate binding induced conformational change before chemistry. In contrast, the incorporation of the next correct triphosphate, dCTP, on a mismatched DNA substrate was saturable with a Km of 87 microM for dCTP, 4-fold higher than the Kd for the correct incorporation on duplex DNA, and a kcat of 0.025 s-1. A larger elemental effect of 60, however, suggests a rate-limiting chemistry step. The rate of pyrophosphorolysis on a mismatched 3'-end is undetectable, indicating that pyrophosphorolysis does not play a proofreading role in replication. These results show convincingly that the T7 DNA polymerase discriminates against the incorrect triphosphate by an induced-fit conformational change and that, following misincorporation, the enzyme then selects against the resultant mismatched DNA by a slow, rate-limiting chemistry step, thereby allowing sufficient time for the release of the mismatched DNA from the polymerase active site to be followed by exonucleolytic error correction.