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 #
5R01GM095558-02
Application #
8308382
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
2012-08-01
Budget End
2013-07-31
Support Year
2
Fiscal Year
2012
Total Cost
$279,971
Indirect Cost
$89,971
Name
University of Minnesota Twin Cities
Department
Biochemistry
Type
Schools of Medicine
DUNS #
555917996
City
Minneapolis
State
MN
Country
United States
Zip Code
55455
Shaban, Nadine M; Shi, Ke; Li, Ming et al. (2016) 1.92 Angstrom Zinc-Free APOBEC3F Catalytic Domain Crystal Structure. J Mol Biol 428:2307-16
Chen, Xiang; Randles, Leah; Shi, Ke et al. (2016) Structures of Rpn1 T1:Rad23 and hRpn13:hPLIC2 Reveal Distinct Binding Mechanisms between Substrate Receptors and Shuttle Factors of the Proteasome. Structure 24:1257-70
Shi, Ke; Carpenter, Michael A; Kurahashi, Kayo et al. (2015) Crystal Structure of the DNA Deaminase APOBEC3B Catalytic Domain. J Biol Chem 290:28120-30
Murphy, Mark W; Lee, John K; Rojo, Sandra et al. (2015) An ancient protein-DNA interaction underlying metazoan sex determination. Nat Struct Mol Biol 22:442-51
Cho, Seunghee; Shi, Ke; Seffernick, Jennifer L et al. (2014) Cyanuric acid hydrolase from Azorhizobium caulinodans ORS 571: crystal structure and insights into a new class of Ser-Lys dyad proteins. PLoS One 9:e99349
Shi, Ke; Huang, Wai Mun; Aihara, Hideki (2013) An enzyme-catalyzed multistep DNA refolding mechanism in hairpin telomere formation. PLoS Biol 11:e1001472
Chen, Luan; Shi, Ke; Yin, Zhiqi et al. (2013) Structural asymmetry in the Thermus thermophilus RuvC dimer suggests a basis for sequential strand cleavages during Holliday junction resolution. Nucleic Acids Res 41:648-56
Cho, Seunghee; Shi, Ke; Wackett, Lawrence P et al. (2013) Crystallization and preliminary X-ray diffraction studies of cyanuric acid hydrolase from Azorhizobium caulinodans. Acta Crystallogr Sect F Struct Biol Cryst Commun 69:880-3
Shi, Ke; Kurahashi, Kayo; Gao, Rui et al. (2012) Structural basis for recognition of 5'-phosphotyrosine adducts by Tdp2. Nat Struct Mol Biol 19:1372-7
Huang, Wai Mun; DaGloria, Jeanne; Fox, Heather et al. (2012) Linear chromosome-generating system of Agrobacterium tumefaciens C58: protelomerase generates and protects hairpin ends. J Biol Chem 287:25551-63