ZUO, ZHONGFENG
(2012)
DNA REPLICATION IN ARCHAEA: PRIMING, TRANSFERASE, AND ELONGATION ACTIVITIES.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
We have biochemically characterized the bacterial-like DnaG primase contained within the hyperthermophilic crenarchaeon Sulfolobus solfataricus (Sso) and compared in vitro priming kinetics with those of the eukaryotic-like primase (PriS&L) also found in Sso. SsoDnaG exhibited metal- and temperature-dependent profiles consistent with priming at high temperatures. The distribution of primer products for SsoDnaG was discrete but highly similar to the distribution of primer products produced by the homologous Escherichia coli DnaG. The predominant primer length was 13 bases, although less abundant products of varying sizes are also present. SsoDnaG was found to bind DNA cooperatively as a dimer with a moderate dissociation constant. Mutation of the conserved glutamate in the active site severely inhibited priming activity, showing functional homology with E. coli DnaG. SsoDnaG was also found to have a greater than four-fold faster rate of DNA priming over that of SsoPriS&L under optimal in vitro conditions. The presence of both enzymatically functional primase families in archaea suggests that the DNA priming role may be shared on leading or lagging strands during DNA replication.
DNA replication polymerases have the inherent ability to faithfully copy a DNA template according to Watson Crick base pairing. The primary B-family DNA replication polymerase (Dpo1) in Sso is shown here to possess a remarkable DNA stabilizing ability for maintaining weak base pairing interactions to facilitate primer extension. This thermal stabilization by SsoDpo1 allowed for template-directed synthesis at temperatures more than 30 °C above the melting temperature of naked DNA. Surprisingly, SsoDpo1 also displays a terminal deoxynucleotide transferase (TdT) activity unlike any other B-family DNA polymerases. SsoDpo1 is shown to elongate single stranded DNA in template-dependent and template-independent manners. The multiple activities of this unique B-family DNA polymerase make this enzyme an essential component for DNA replication and DNA repair for the maintenance of the archaeal genome at high temperatures.
Preliminary results of primer transfer studies in Sso show that SsoDpo1 can elongate DNA primers de novo synthesized by SsoPriS&L, but elongation of RNA primers synthesized by both SsoPriS&L and SsoDnaG was not observed for SsoDpo1. Future studies to untangle the primer transfer mechanism in archaea are discussed.
Share
| Citation/Export: |
|
| Social Networking: |
|
Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
|
| ETD Committee: |
|
| Date: |
5 July 2012 |
| Date Type: |
Publication |
| Defense Date: |
27 January 2012 |
| Approval Date: |
5 July 2012 |
| Submission Date: |
30 March 2012 |
| Access Restriction: |
No restriction; Release the ETD for access worldwide immediately. |
| Number of Pages: |
134 |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
Dietrich School of Arts and Sciences > Chemistry |
| Degree: |
PhD - Doctor of Philosophy |
| Thesis Type: |
Doctoral Dissertation |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
ARCHAEA, DNA PRIMASE, DNA POLYMERASE, DNA TERMINAL TRANSFERASE, SULFOLOBUS SOLFATARICUS |
| Date Deposited: |
05 Jul 2012 18:51 |
| Last Modified: |
15 Nov 2016 13:57 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/11631 |
Metrics
Monthly Views for the past 3 years
Plum Analytics
Actions (login required)
 |
View Item |
![[feed]](/images/feed-icon-32x32.png){kind=icon}