Abstract
Extension of synthetic primers by purified human polymerase alpha (Hpol alpha) with the (+)-strand of M13mp18 DNA as template encounters numerous specific pause sites on the M13 template. Some of these are regions of template secondary structure, at others the template codes for incorporation of the same base in multiple consecutive positions, but at some the responsible feature in the sequence is not obvious. 2-Chloro-dATP (CldATP) substitutes efficiently for dATP in such chain extension, with 2-chloroadenine (ClA) incorporation into many positions coding for A. However, there are more sites where extension is interrupted than with all four normal nucleotide substrates, particularly (but not exclusively) at template secondary structure and sites of multiple consecutive ClA insertion. DNA synthesis from normal substrates by Hpol beta in this system shows less frequent and less marked pauses, but with CldATP substituted for dATP chain extension is limited because of marked slowing of extension at sites of multiple consecutive ClA insertion. With either polymerase, the rate of extension is decreased even more at such regions when bromo-dATP is used as substrate. Some misincorporation of ClA instead of G or T can occur at certain sites in absence of the corresponding normal substrate, but misincorporation as C is rare. CldATP is a very weak inhibitor of chain extension by Hpol alpha, but a somewhat better inhibitor of Hpol beta. These results may account in part for the inhibition of DNA synthesis in cells exposed to 2-chlorodeoxyadenosine or 2-bromodeoxyadenosine.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.