scholarly journals MTBP, the partner of Treslin, contains a novel DNA-binding domain that is essential for proper initiation of DNA replication

2017 ◽  
Vol 28 (22) ◽  
pp. 2998-3012 ◽  
Author(s):  
Akiko Kumagai ◽  
William G. Dunphy
Keyword(s):  
Dna Replication ◽  
Critical Role ◽  
Replicative Helicase ◽  
Double Stranded Dna ◽  
G4 Dna ◽  
G Quadruplex ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Initiation Of Dna Replication ◽  
Xenopus Egg Extracts

Treslin, which is essential for incorporation of Cdc45 into the replicative helicase, possesses a partner called MTBP (Mdm2-binding protein). We have analyzed Xenopus and human MTBP to assess its role in DNA replication. Depletion of MTBP from Xenopus egg extracts, which also removes Treslin, abolishes DNA replication. These extracts be can rescued with recombinant Treslin-MTBP but not Treslin or MTBP alone. Thus, Treslin-MTBP is collectively necessary for replication. We have identified a C-terminal region of MTBP (the CTM domain) that binds efficiently to both double-stranded DNA and G-quadruplex (G4) DNA. This domain also exhibits homology with budding yeast Sld7. Mutants of MTBP without a functional CTM domain are defective for DNA replication in Xenopus egg extracts. These mutants display an impaired localization to chromatin and the inability to support loading of Cdc45. Human cells harboring such a mutant also display severe S-phase defects. Thus, the CTM domain of MTBP plays a critical role in localizing Treslin-MTBP to the replication apparatus for initiation.

scholarly journals Replication Fork Uncoupling Causes Nascent Strand Degradation and Fork Reversal

2021 ◽  
Author(s):  
Tamar Kavlashvili ◽  
James M Dewar
Keyword(s):  
Dna Replication ◽  
Replication Fork ◽  
Genome Stability ◽  
Replicative Helicase ◽  
Before And After ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Biochemical Approach ◽  
Key Steps ◽  
Xenopus Egg Extracts

Genotoxins cause nascent strand degradation (NSD) and fork reversal during DNA replication. NSD and fork reversal are crucial for genome stability and exploited by chemotherapeutic approaches. However, it is unclear how NSD and fork reversal are triggered. Additionally, the fate of the replicative helicase during these processes is unknown. We developed a biochemical approach to study synchronous, localized NSD and fork reversal using Xenopus egg extracts. We show that replication fork uncoupling stimulates NSD of both nascent strands and progressive conversion of uncoupled forks to reversed forks. The replicative helicase remains bound during NSD and fork reversal, indicating that both processes take place behind the helicase. Unexpectedly, NSD occurs before and after fork reversal, indicating that multiple degradation steps take place. Overall, our data show that uncoupling causes NSD and fork reversal and identify key steps involved in these processes.

scholarly journals Cdc7-Drf1 kinase links chromosome cohesion to the initiation of DNA replication in Xenopus egg extracts

2008 ◽  
Vol 22 (14) ◽  
pp. 1894-1905 ◽  
Author(s):  
T. S. Takahashi ◽  
A. Basu ◽  
V. Bermudez ◽  
J. Hurwitz ◽  
J. C. Walter
Keyword(s):  
Dna Replication ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Initiation Of Dna Replication ◽  
Xenopus Egg Extracts
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