Initiation Of Dna Replication In Mammalian Chromosomes

哺乳动物染色体中 DNA 复制的起始

• 批准号: 6681730
• 负责人: Melvin DePamphilis
• 金额: --
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6681730
• 关键词: DNA directed DNA polymerase; DNA replication; DNA replication origin; chromosomes; enzyme activity; genetic regulatory element; hamsters; nucleic acid sequence; tissue /cell culture

We have focused on those aspects of eukaryotic DNA replication that appear to be unique to the metazoa: how do the metazoa regulate the number and locations of initiation sites during cell proliferation and development? We have previously mapped origins of bi-directional replication (OBRs) to specific chromosomal sites of 0.4 to 2 kb in the hamster genome. We have now been able to demonstrate that pre-replication complexes are assembled at or close to these sites when hamster cells transit from metaphase to G1 phase in their cell division cycle. These pre-RCs can be activated in vitro using a Xenopus egg extract. In eukaryotes, site specificity is determined by a six member ?origin recognition complex? that binds to specific sites along the genome. Fission yeast (S. pombe), like mammals, contain large, AT-rich replication origins that lack a recognizable consensus sequence, but that nevertheless contain sequences required for replicatioin. We have discovered that the SpOrc4p alone bound tightly and specifically to several sites within S. pombe replication origins that are genetically required for origin activity. These sites consisted of clusters of A or T residues on one strand, but were devoid of either alternating A and T residues or GC-rich sequences. Addition of a complex consisting of Orc1, 2, 3, 5 and 6 proteins (ORC-5) did not alter either Orc4p binding to origin DNA, or Orc4p protection of specific sequences. ORC-5 alone bound weakly and non-specifically to DNA; strong binding required the presence of Orc4p. Under these conditions, all six subunits remained bound to chromatin isolated from each phase of the cell division cycle. These results reveal that the S. pombe ORC binds to multiple, specific sites within replication origins, and that site selection, at least in vitro, is determined solely by the Orc4p subunit. We have further shown that Orc4p binds specifically to only one of the four required sequences in ARS3001 where it initiates assembly of a pre-RC and initiates bi-directional DNA replication. Thus, S. pombe replication origins may provide an appropriate paradigm for replication origins in higher eukaryotes. In mammals, we have identified the existence of a novel regulatory pathway in the initiation of DNA replication. Orc1 and Orc2 are both tightly bound to chromatin during late G1?phase, and late G1?phase nuclei contain active ORC/chromatin sites by virtue of the fact that they can initiate DNA replication at specific genomic sites when incubated in an Orc?depleted Xenopus egg extract. In contrast to yeast where all six ORC subunits are stably bound to chromatin throughout the cell cycle, the affinity of mammalian Orc1 for chromatin is selectively reduced during S-phase, such that lysis of cells in 0.1% Triton X-100, 0.15 M NaCl, and 1 mM Mg++ATP releases Orc1, but not Orc2, into the chromatin unbound fraction. Moreover, the Orc1 that is released during S-phase is rapidly ubiquitinated with only one or two ubiquitin adducts. In contrast, Orc2 is not a substrate for ubiquitination. During the S to M transition, Orc1 is deubiquitinated. During the M to G1 transition, Orc1 rebinds tightly to hamster ORC/chromatin sites to allow assembly of pre-replication complexes. This sites are located at specific genomic loci referred to as ?origins of bi-directional replication?. The role of Ubiquitination is to sequester Orc1 during S-phase, and thus prevent reinitiation at replication origins during a single cell division cycle. However, if Ub-Orc1 is released into the cytosol, then it is polyubiquitinated and degraded by the 26S proteasome pathway. This provides a mechanism for reprogramming replication origins during animal development or as a result of DNA damage. Thus, in contrast to yeast, mammalian ORC activity appears to be regulated during each cell cycle through selective dissociation and reassociation of Orc1 from chromatin bound ORC. In searching for the trigger that releases Orc1 from mammalian chromatin, we incubated mammalian mitotic chromatin (which lacks functional ORCs) in a Xenopus egg extract to initiate DNA replication. We discovered that the entire Xenopus ORC rapidly binds to the somatic cell chromatin, initiates DNA replication, and is released as soon as Mcm proteins are bound to form a pre-RC.

我们关注的是真核DNA复制的那些方面，这些复制似乎是Metazoa所独有的：如何在细胞增殖和发育过程中调节起始位点的数量和位置？以前，我们已经映射了双向复制（OBR）的起源，以在仓鼠基因组中为0.4至2 kb的特定染色体位点。现在，当仓鼠细胞在其细胞分裂周期中从中期转移到G1相时，我们已经能够证明，预复合复合物在这些位点或接近这些位点组装。这些PER-RC可以在体外使用异武鸡蛋提取物在体外激活。 在真核生物中，场地特异性由六个成员？原点识别综合体确定？与沿基因组的特定位点结合。裂变酵母（S. pombe）像哺乳动物一样，包含缺乏可识别的共识序列的大型，富集的复制起源，但仍然包含复制蛋白所需的序列。我们已经发现，单独的SPORC4P仅紧密地绑定到S. pombe复制起源中的几个位点，这些位点是原始活动所需的。这些位点由一条链上的A或T残基组成，但没有交替的A和T残基或富含GC的序列。添加由ORC1、2、3、5和6蛋白（ORC-5）组成的复合物不会改变与原点DNA的ORC4P结合，或者对特定序列的ORC4P保护。 ORC-5单独与DNA薄弱地结合；强大的结合需要存在ORC4P。在这些条件下，所有六个亚基仍然与从细胞分裂周期的每个阶段分离的染色质绑定在一起。这些结果表明，pombe S. orc与复制起源中的多个特定位点结合，至少在体外的位点选择仅由ORC4P亚基确定。我们进一步表明，ORC4P专门与ARS3001中的四个必需序列之一进行结合，在ARS3001中它启动了PRE-RC的组装并启动双向DNA复制。因此，S。pombe复制起源可以为较高的真核生物中的复制起源提供适当的范式。 在哺乳动物中，我们已经确定了在DNA复制启动时存在新的调节途径。 ORC1和ORC2在G1后期都与染色质紧密结合，并且G1？阶段核含有活跃的ORC/染色质位点，这是因为它们可以在兽人中孵育时可以在特定的基因组位点启动DNA复制。 In contrast to yeast where all six ORC subunits are stably bound to chromatin throughout the cell cycle, the affinity of mammalian Orc1 for chromatin is selectively reduced during S-phase, such that lysis of cells in 0.1% Triton X-100, 0.15 M NaCl, and 1 mM Mg++ATP releases Orc1, but not Orc2, into the chromatin unbound fraction.此外，在S期期间释放的ORC1仅用一个或两个泛素加合物迅速泛素化。相反，ORC2不是泛素化的底物。在s到m过渡期间，ORC1被去泛素化。在M到G1过渡期间，ORC1紧紧地重新启动到仓鼠兽/染色质位点，以允许组装预复合物。该站点位于特定的基因组基因座，称为双向复制的起源？泛素化的作用是在S期间隔离ORC1，从而防止在单个细胞分裂周期中复制起源时重新定期。但是，如果UB-ORC1被释放到细胞质中，则将其由26S蛋白酶体途径进行多泛素化和降解。这提供了一种在动物发育期间或DNA损伤导致重新编程复制起源的机制。因此，与酵母相比，在每个细胞周期中，通过选择性解离和重新关联ORC1从染色质型ORC中，在每个细胞周期中似乎都受到调节。 在搜索从哺乳动物染色质释放ORC1的触发器时，我们在Xenopus鸡蛋提取物中孵育哺乳动物有丝分裂染色质（缺乏功能性ORC）以启动DNA复制。我们发现，整个爪蟾兽人迅速结合与体细胞染色质，启动DNA复制，并在MCM蛋白绑定形成前RC后立即释放。