Physical Biochemistry Of Macromolecules

与塞缪尔·威尔逊(NIEHS)博士的实验室就参与DNA转录启动和DNA修复的蛋白质进行的合作研究仍在继续。这些工作包括DNA连接酶I与复制蛋白、增殖细胞核抗原的相互作用；XRCC1(1-183)蛋白与DNA聚合酶-β及其亚域的相互作用机制；AP内切酶与DNA、DNA聚合酶-β与DNA的相互作用，以及两者与DNA的相互作用。在DNA酶的研究方面，已完成了两个方面的研究，一是增殖细胞核抗原齐聚的热力学，二是增殖细胞核抗原与组氨酸标记和未标记的FEN-1的相互作用。两份手稿正在准备中。预计这项工作将在来年继续进行。 在与Carl Wu(NCI)博士的实验室合作的研究中，我们证明了蛋白质Chz1、Htz1和H2B形成了1：1：1化学计量比的可逆复合体。这种复合体似乎是一种新的组蛋白伴侣，对H_2AZ具有特异性，并可以在真核细胞中递送组蛋白变体用于SWR1依赖的组蛋白替代。近日，《分子细胞》杂志上发表了一篇题为《Chz1，一种新型的组蛋白H_2AZ核伴侣》的手稿。 在与Daniel Appella博士(NIDDK)的一项合作研究中，我们一直在研究人工合成的寡核苷酸(这里称为DNA)以及与多肽骨架具有相同碱基的类似分子(此处称为PNA)的自结合特性。我们已经证明了DNA具有极高的热稳定性，并且经历了可逆的二聚体-四聚体自缔合，其平均自由能变化为-6.88千卡/摩尔，与温度的依赖性很小，并且几乎完全来自于热源。这与其结构完全一致。我们发现PNA的稳定性明显低于DNA，在34℃时显示出降解的迹象。C.它经历了单体-四聚体的自缔合，平均自由能变化为-7.7千卡/摩尔，比DNA表现出的自缔合更弱，因为这是二聚体-四聚体缔合，而PNA经历的是单体-四聚体缔合。PNA的自缔合行为对温度的依赖性很小，而且几乎完全是热源的，这也与它的结构一致。DNA和PNA的混合物与-19.6千卡/摩尔的平均自由能变化密切相关。然而，它表现出不同寻常的温度依赖性，初步分析给出了热力学参数的意想值。自由能变化的平均值为-3.4千卡/摩尔，平均变化的熵为59千卡/摩尔/K，比热含量的平均值为-2.5千卡/摩尔/K。因此，自由能变化的值本质上是一种热熵补偿。虽然这在蛋白质-蛋白质关联中很常见，但在这里是相当出乎意料的，需要相当多的额外研究来解释。这与DNA或PNA的热力学行为形成鲜明对比。此外，在28℃的温度下。C及以上，未检测到异四聚体。这是由于PNA的降解，还是由于形成了异四聚体以外的其他结构，这一点还有待确定。这一问题具有相当大的生物学意义，因为PNA似乎是抑制DNA转录的一种特别特异的手段，并可能在控制恶性肿瘤方面发挥重要作用。 几年前与NCI的Ettore Appella博士的实验室合作的结果，题为“与肺癌相关的突变型p53的四聚体结构域的展开、聚集和淀粉样形成”，已发表在《生物化学》杂志上。

Collaborative studies with the laboratory of Dr. Samuel Wilson (NIEHS) on proteins involved in DNA transcription initiation and in DNA repair have continued. These have included work on the interactions between DNA Ligase I and the replication protein, proliferating cell nuclear antigen (PCNA); the interaction mechanisms between the XRCC1(1-183) protein and DNA polymerase-beta and its subdomains; the interactions between AP endonuclease and DNA, DNA polymerase-beta and DNA, and both together with DNA. Research has been completed on two aspects of the studies on DNA enzymes, the thermodynamics of oligomerization of PCNA and the interaction of PCNA with histidine-tagged and untagged FEN-1. Two manuscripts are in preparation. It is anticipated that this work will continue in the coming year. In a collaborative study with the laboratory of Dr. Carl Wu (NCI), we have demonstrated that the proteins Chz1, Htz1, and H2B form a reversible complex with a 1:1:1 stoichiometry. This complex appears to be a novel histone chaperone that has specificity for H2AZ and can deliver the histone variant for SWR1-dependent histone replacement in eukaryotic cells. A manuscript entitled "Chz1, a Novel Nuclear Chaperone for Histone H2AZ" has been published in the journal "Molecular Cell." In a collaborative study with the laboratory of Dr. Daniel Appella (NIDDK), we have been studying the self-associative properties of a synthetic oligonucleotide (referred to here as DNA) and a similar molecule with the same bases attached to a polypepetide backbone (referred to here as PNA). We have demonstrated that the DNA is extremely thermally stable and undergoes a reversible dimer-tetramer self-association with a mean free energy change of -6.88 kcal/mol that has very little temperature dependence and is virtually totally of enthalpic origin. This is entirely consistent with its structure. We find that the PNA has significantly less stability than the DNA, showing signs of degradation at 34 deg. C. It undergoes a monomer-tetramer self-association with a mean free energy change of -7.7 kcal/mol, a weaker self-association than that exhibited by the DNA, since that is for a dimer-tetramer association and the PNA undergoes a monomer-tetramer association. The PNA self-association exhibits little temperature dependence and also is virtually totally of enthalpic origin, again consistent with its structure. A mixture of the DNA and the PNA associates very strongly with a mean free energy change of -19.6 kcal/mol. However, it exhibits unusual temperature dependency and preliminary analysis gave unexpected values for the thermodynamic parameters. The mean value of the change in enthalpy was -3.4 kcal/mol; the mean value for the change in entropy was 59 cal/mol/K; and the mean value for the specific heat content was -2.5 kcal/mol/K. Thus, the values of the change in free energy appear to be essentially a case of enthalpy-entropy compensation. While this is quite common in protein-protein association, it is quite unexpected here and will require considerable additional studies to explain. This is in marked contrast to the thermodynamic behavior of either the DNA or the PNA. Additionally, at temperatures of 28 deg. C and above, no heterotetramer was detectable. Whether this is due to the initiation of degradation of the PNA or the formation of some structure other than heterotetramer remains to be determined. This problem is of considerable biological interest since the PNAs appear to be a particularly specific means for the inhibition of DNA transcription and may have a significant role in the control of malignancy. The results of a collaboration with the laboratory of Dr. Ettore Appella (NCI), performed several years ago, entitled "Unfolding, Aggregation and Amyloid Formation by the Tetramerization Domain From Mutant p53 Associated With Lung Cancer" has been published in the journal "Biochemistry."