Telomeres protect the ends of a linear chromosome and facilitate complete replication of terminal DNA sequences. The simplest form of telomere is a covalently closed hairpin structure found in bacteria and viruses carrying linear chromosomes, including members of the genus Borrelia - the causative agents of Lyme disease and relapsing fever - and the poxviruses. Replication of a linear chromosome with hairpin telomeres proceeds through a two-step mechanism, in which DNA polymerases first produce a concatenated replication intermediate that is subsequently resolved into unit-length chromosomes. This proposal focuses on the bacterial and poxviral enzymes that resolve the concatemeric chromosome to regenerate hairpin telomeres. The two classes of DNA resolvases utilize distinct types of chemical reactions to process the inverted repeat DNA sequences separating multiple copies of chromosomes. Bacterial protelomerases resolve a palindromic duplex substrate into hairpin products using the phophotyrosine-mediated DNA cleavage-rejoining reaction. On the other hand, the poxvirus resolvase binds specifically to the Holliday junction structure formed by hairpin extrusion at a palindromic sequence and makes symmetrical strand cleavages across the junction point. Even though the chemical natures of the reactions catalyzed by the bacterial and poxviral DNA resolvases are well established, it is poorly understood how these proteins adapt the respective catalytic modules to resolve DNA at the sites of replicated telomeres. In this proposal we will specifically address the following questions by determining crystal structures of various resolvase- DNA complexes: How do the bacterial protelomerase enzymes facilitate efficient refolding of a duplex substrate into hairpin products using the intrinsically isoenergetic DNA cleavage-rejoining chemistry? How does the poxvirus resolvase achieve high specificity in recognizing the branched DNA structure and catalyzing concerted strand cleavages at the junction point? Despite carrying out reactions seemingly independent of each other at the biochemical level, the two types of DNA resolvases may share a similar strategy in making symmetrical DNA cleavages across the inverted repeat junction. Our structural work will highlight diverse strategies as well as potentially a general mechanism employed by enzymes involved in the maintenance of hairpin telomeres in the important pathogens. Furthermore, our research may contribute to better understanding of many DNA rearrangement machineries that share similar reaction chemistries with the DNA resolvases studied here.

Public Health Relevance

We will investigate how the genetic information is maintained and replicated in certain bacteria and viruses that cause human diseases or problems in food industry. The information obtained through this research will help design drugs, and may contribute to development of tools for manipulating DNA in a wide range of applications.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM095558-03
Application #
8513361
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Preusch, Peter C
Project Start
2011-08-01
Project End
2016-07-31
Budget Start
2013-08-01
Budget End
2014-07-31
Support Year
3
Fiscal Year
2013
Total Cost
$270,172
Indirect Cost
$86,822
Name
University of Minnesota Twin Cities
Department
Biochemistry
Type
Schools of Medicine
DUNS #
555917996
City
Minneapolis
State
MN
Country
United States
Zip Code
55455