Caracterización del mecanismo de acción de la helicasa replicativa G40P y su cargador G39P en la replicación del bacteriófago SPP1 de Bacillus subtilis
Author
Mesa García, PabloAdvisor
Alonso Navarro, Juan CarlosEntity
UAM. Departamento de Biología MolecularDate
2014Subjects
Bacteriófagos - Tesis doctorales; Bacilus subtilis - Tesis doctorales; Biología y Biomedicina / BiologíaNote
Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 2014Esta obra está bajo una licencia de Creative Commons Reconocimiento-NoComercial-SinObraDerivada 4.0 Internacional.
Abstract
The Bacillus subtilis SPP1-encoded G40P is a hexameric replicative helicase that
unwinds dsDNA with a 5’!3’ polarity. During the amplification of the SPP1 genome this
DnaB-like helicase uses its ATP-powered motor activity to provide ssDNA to the host
polymerases, the DnaG primase and the DNA polymerases (polC and DnaE). G40P is also
involved in the initiation of SPP1 DNA replication, with two other phage-encoded proteins:
G39P and G38P. Through its interactions with G40P and G38P, G39P is able to load the
helicase into the replication origin (oriL), which is recognized and locally unwound by G38P.
This system represents an alternative mechanism to the reference model exemplified by
Escherichia coli DNA replication initiation proteins: DnaA, DnaC and DnaB.
This work addresses the study of G40P helicase activity and the role of G39P in the
helicase loading mechanism. Different deletion constructs were used in order to link functions
to structural features of these two proteins. In this regard, a G40P mutant lacking the N-terminal
domain, G40PΔN109, showed two unexpected activities. It was able to unwinds DNA
bidirectionally, with an additional 3’!5’ translocation activity which is absent in wild-type
G40P. Surprisingly, G40PΔN109 was also capable of anneal complementary DNA strands. In
order to explain these activities, we propose a regulatory function for the N-terminal domain of
G40P. This part of the helicase could be involved in the control of the relative disposition of the
ATPase C-terminal domains and in the regulation of the DNA-binding activity. Structural and
biochemical studies showed that G39P contains two different domains, a folded N-terminal
domain and a natively disordered C-terminal domain. We propose that this bimodular structure
is essential for G39P to function as a helicase loader. The interaction of the disordered regions
with G40P would induce the association of the N-terminal domains of G39P, and this event
could force the opening of the helicase ring. Then G40P is loaded on the DNA and G39P is
released. By using this mechanism G39P promotes the loading of G40P on ssDNA and prevents
the loading on dsDNA.
In addition, small natural compounds griseorhodin C and purpuromycin were found to
inhibit the ATPase and helicase activities of G40P.
Files in this item
Google Scholar:Mesa García, Pablo
This item appears in the following Collection(s)
Related items
Showing items related by title, author, creator and subject.
-
Purificación y Caracterización del Mecanismo de Acción de la Proteína RecN de Bacillus subtilis
Sabariegos Jareño, María del Rosario
1999-01-28