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Incorporation of 2-halogeno-2'-deoxyadenosine 5-triphosphates into DNA during replication by human polymerases alpha
P Hentosh1, R Koob, R L Blakley
1Department of Biochemical and Clinical Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101.
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.
Insights
Modified nucleotides like 2-chloro-dATP (CldATP) can halt DNA synthesis by human DNA polymerases (Hpol alpha and Hpol beta). These pauses, especially at specific DNA sequences, may explain how related compounds inhibit cellular DNA replication.
Area of Science:
- Molecular Biology
- Biochemistry
Background:
- DNA polymerases synthesize DNA by extending primers along a template strand.
- Specific DNA sequences and template secondary structures can cause DNA polymerase pausing during synthesis.
- Modified nucleotides are used to study DNA synthesis mechanisms and potential therapeutic applications.
Purpose of the Study:
- To investigate the effects of 2-chloro-dATP (CldATP) and 2-bromo-dATP on DNA synthesis by human DNA polymerase alpha (Hpol alpha) and human DNA polymerase beta (Hpol beta).
- To identify sequence-specific pause sites encountered during DNA synthesis with modified nucleotides.
- To explore the potential role of these modified nucleotides in inhibiting cellular DNA synthesis.
Main Methods:
- In vitro DNA synthesis assays using M13mp18 DNA as a template.
- Extension of synthetic primers by purified Hpol alpha and Hpol beta.
- Substitution of dATP with CldATP or bromo-dATP in the reaction mixtures.
- Analysis of pause sites and misincorporation events during DNA chain extension.
Main Results:
- Hpol alpha encounters numerous pause sites with normal substrates, exacerbated by CldATP, especially at template secondary structures and multiple consecutive CldA incorporation sites.
- Hpol beta shows fewer pauses with normal substrates, but CldATP significantly slows extension at multiple consecutive ClA insertion sites.
- Bromo-dATP further decreases extension rates at these problematic regions for both polymerases.
- CldA misincorporation occurs at specific sites, but is rare as C. CldATP weakly inhibits Hpol alpha but moderately inhibits Hpol beta.
Conclusions:
- CldATP and bromo-dATP can cause significant pausing and inhibition of DNA synthesis by human DNA polymerases.
- The observed pausing is sequence-dependent and influenced by template secondary structures and repetitive nucleotide incorporation.
- These findings provide a mechanistic basis for the inhibition of DNA synthesis in cells treated with 2-chlorodeoxyadenosine or 2-bromodeoxyadenosine.