机械洞察AP-endonuclease 1在停滞的复制叉上模仿基基部位的切割
Nicole M Hoitsma1, Jessica Norris2, Thu H Khoang1
1Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, KS 66160, USA.
Nucleic acids research
|June 2, 2023
概括
AP内核酶1 (APE1) 在复制分叉处有效地处理单链DNA (ssDNA) 中的基基位,即使与复制蛋白A (RPA) 结合. 这揭示了对DNA修复机制和复制叉稳定性的关键分子见解.
科学领域:
- 分子生物学分子生物学
- DNA 修复机制的修复机制
- 结构生物学 结构生物学
背景情况:
- 亚基位等DNA损伤可以阻断DNA复制叉,导致复制压力和单链DNA (ssDNA) 的生成.
- 众所周知,AP内核酶1 (APE1) 处理基底部位,但其在复制分叉中的作用和效率,特别是在ssDNA上,尚未完全理解.
- 复制蛋白A (RPA) 是一种关键的ssDNA结合蛋白,在复制分叉时覆盖暴露的ssDNA.
研究的目的:
- 调查APE1在ssDNA基底内处理基点的分子基础和效率,以模仿停滞的复制分叉或间隙.
- 阐明APE1在ssDNA上的活动背后的结构机制.
- 检查在复制分叉的基础站点处理过程中APE1和RPA之间的相互作用.
主要方法:
- 生物化学测试以确定APE1在基底SSDNA基板上的裂变速率和产品释放动力学.
- 进行X射线晶体学以可视化与ssDNA基质相互作用的APE1活性位点.
- 位点定向突变发生,以识别关键残留物,如R177,参与APE1的ssDNA裂变机制.
- 与APE1,RPA和底层ssDNA基质进行共同化实验.
主要成果:
- APE1在底层ssDNA基板上表现出强大的裂变活性,与其在双链DNA (dsDNA) 上的活性相当.
- X射线结构显示,APE1在ssDNA处理和dsDNA处理中使用类似的活性位点机制,其残留物R177对ssDNA裂变具有独特的关键性.
- 即使RPA与ssDNA结合在一起,APE1也可以有效地切割基底部部位,这表明这些蛋白质之间的功能相互作用.
结论:
- APE1在复制分叉处高效地处理ssDNA中的基点,有助于解决复制压力.
- 结构和突变分析为APE1的ssDNA裂变机制提供了分子洞察力,突出了R177.7的特定作用.
- 在RPA的存在下APE1的功能能力表明,在停滞不前的复制分叉上有协调的DNA修复过程.
相关概念视频
Restarting Stalled Replication Forks
5.8K
DNA replication is initiated at sites containing predefined DNA sequences known as origins of replication. DNA is unwound at these sites by the minichromosome maintenance (MCM) helicase and other factors such as Cdc45 and the associated GINS complex.The unwound single strands are protected by replication protein A (RPA) until DNA polymerase starts synthesizing DNA at the 5’ end of the strand in the same direction as the replication fork. To prevent the replication fork from falling apart,...
5.8K
Homologous Recombination
50.7K
The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One of these sequences uses a strand from another as a template to synthesize DNA in an enzyme-catalyzed reaction. The final product is a novel amalgamation of the two substrates. To ensure an accurate recombination of sequences, HR is restricted to the S and G2 phases of the cell cycle. At these stages, the DNA has been replicated already and the...
50.7K
The DNA Replication Fork
36.2K
An organism’s genome needs to be duplicated in an efficient and error-free manner for its growth and survival. The replication fork is a Y-shaped active region where two strands of DNA are separated and replicated continuously. The coupling of DNA unzipping and complementary strand synthesis is a characteristic feature of a replication fork. Organisms with small circular DNA, such as E. coli, often have a single origin of replication; therefore, they have only two replication...
36.2K
Lagging Strand Synthesis
53.5K
During replication, the complementary strands in double-stranded DNA are synthesized at different rates. Replication first begins on the leading strand. Replication starts later, occurs more slowly, and proceeds discontinuously on the lagging strand.
There are several major differences between synthesis of the leading strand and synthesis of the lagging strand. 1) Leading strand synthesis happens in the direction of replication fork opening, whereas lagging strand synthesis happens in the...
There are several major differences between synthesis of the leading strand and synthesis of the lagging strand. 1) Leading strand synthesis happens in the direction of replication fork opening, whereas lagging strand synthesis happens in the...
53.5K
Translesion DNA Polymerases
10.1K
Translesion (TLS) polymerases rescue stalled DNA polymerases at sites of damaged bases by replacing the replicative polymerase and installing a nucleotide across the damaged site. Doing so, TLS allows additional time for the cell to repair the damage before resuming regular DNA replication.
TLS polymerases are found in all three domains of life - archaea, bacteria, and eukaryotes. Of the different classes of TLS polymerases, members of the Y family are fitted with specialized structures that...
TLS polymerases are found in all three domains of life - archaea, bacteria, and eukaryotes. Of the different classes of TLS polymerases, members of the Y family are fitted with specialized structures that...
10.1K
DNA Damage can Stall the Cell Cycle
9.2K
In response to DNA damage, cells can pause the cell cycle to assess and repair the breaks. However, the cell must check the DNA at certain critical stages during the cell cycle. If the cell cycle pauses before DNA replication, the cells will contain twice the amount of DNA. On the other hand, if cells arrest after DNA replication but before mitosis, they will contain four times the normal amount of DNA. With a host of specialized proteins at their disposal,cells must use the right protein at...
9.2K


