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Sinigalia, Elisa (2008) Caratterizzazione funzionale e ricerca di partners proteici cellulari della subunità accessoria, UL44, della DNA polimerasi del citomegalovirus umano. [Ph.D. thesis]

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Abstract (english)

The human cytomegalovirus (HCMV) DNA polymerase consists of a catalytic subunit, UL54, and an accessory protein, UL44, that is thought to act as a processivity factor. UL44 has been shown to bind double-stranded DNA, to specifically interact with UL54 and to stimulate long-chain DNA synthesis by UL54.
The crystal structure of UL44 has recently been solved and has revealed that UL44 possesses a structural fold similar to that of other processivity factors, including herpes simplex virus UL42 and the eukaryotic sliding clamp PCNA. It has also been shown that UL44 forms a homodimer. Similarly to UL42, the putative DNA-binding face of UL44 contains multiple basic residues that could interact electrostatically with the phosphate backbone of DNA.
Residues important for the dimerization of UL44 have been identified: L86, L87 and F121. Indeed, the F121A and L86A/L87A mutants, which behave like monomers, are impaired in the ability to bind double-strand DNA.
In this work we performed some experiments to better understand the role of UL44 dimerization in vivo. We demonstrated that, as already shown for truncated UL44?C290, full-length UL44 can form dimers both in solution and in a cellular context. Moreover, while UL44 dimerization has been shown to be important for binding to DNA, it appears to be not required for binding to UL54 and for stimulation of DNA synthesis by UL54 in vitro. We also investigated the effect of the L86A/L87A and F121A mutations of UL44 on viral DNA replication through an in vivo assay. The homodimerization of UL44 turned out to be essential for the replication of the viral DNA in a cellular context. This observation suggests that the disruption of UL44 dimerization could represent a good strategy for the development of new antiviral compounds.
How UL44 binds to DNA and the role of DNA binding in processivity function have not been yet elucidated. To begin to understand these mechanism, we characterized the interaction of UL44 with DNA by means of filter-binding assays and electrophoretic mobility shift assays (EMSA). We found that, similar to HSV-1 UL42, UL44 binds directly to DNA with nanomolar affinity in a manner that does not require ATP hydrolysis or accessory proteins. UL44 binds DNA as a dimer in a sequence-non specific manner and displays higher affinity for ds DNA compared to ss DNA. Affinity of UL44 for ds DNA decreases with increasing ionic strength and this effect is mediated by ion release, suggesting that DNA binding entails electrostatic interactions between the negatively charged phosphates on DNA backbone and the positive charge of basic residues on the ‚Äúback‚ÄĚ face and disordered loops of UL44.
Finally, several observations suggest that UL44 could have other function(s) besides that of DNA processivity factor. Hence, we performed a two-hybrid system screening in order to identify cellular protein partners of UL44 using a cellular cDNA library. We were able to identify 7 cellular proteins which could represent UL44 functional partners.

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EPrint type:Ph.D. thesis
Tutor:Loregian, Arianna
Ph.D. course:Ciclo 20 > Corsi per il 20simo ciclo > VIROLOGIA E BIOTECNOLOGIE MICROBICHE
Data di deposito della tesi:January 2008
Anno di Pubblicazione:January 2008
Key Words:HCMV, DNA polimerasi, EMSA, sistema del doppio ibrido, GST-pulldown
Settori scientifico-disciplinari MIUR:Area 06 - Scienze mediche > MED/07 Microbiologia e microbiologia clinica
Struttura di riferimento:Dipartimenti > pre 2012 Dipartimento di Istologia, Microbiologia e Biotecnologie Mediche
Codice ID:377
Depositato il:11 Nov 2008
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