INTEGRATED RESEARCH ASSINGMENT DNA helicase-DNA (dnaB)

DNA helicase; are a class of enzymes that are molecular motor proteins that harness the energy of
ATP hydrolysis to catalyse the unwinding of DNA (Rasnik et al, 2002). DNA helicases have been
separated into six major superfamilies based on the consensus sequences shared by the molecules
(Tuteja and Tuteja, 2004). DNA helicases are also classified as alpha or beta; alpha DNA helicase
interacts with ssDNA and beta DNA helicase interacts with dsDNA (Rasnik et al, 2002). The Bacillus
subtilis bacteriophage SPP1 helicase, G40P, is a homolog of bacterial DnaB helicase (Wang et al,
2008). G40P has the same domain structure as other bacterial DnaB homologs (Wang et al, 2008).
The crystal structure of G40P revealed two distinct monomer conformations; cis and trans, which
alternate and assemble into one hexamer. There is a double tiered structure in the molecule; a
three-fold N-terminal tier and a six-fold C-terminal ATPase tier in the hexamer (Wang et al, 2008).
The N-terminal domains are important for helicase function and contain the dnaG binding sites. The
dnaG binding sites are comprised of; an N-globe and alpha-hairpin structures. The monomer
structure of G40P is made up of three domains; an N-terminal (residues 1293) which consists of
four alpha-helices, a 'linker' region (residues 94147) composed of two long alpha-helices, and a C-
terminal RecA-like domain (residues 179437) (Wang et al, 2008).

















DNA; is a molecule that encodes genetic information used in the development and function of all
known living organisms. DNA is a double stranded polynucleotide molecule, with each nucleotide
being composed of a base, which is either; guanine (G), adenine (A), thymine (T), or cytosine (C)
(Watson and Crick, 1953). The base is also bound to a sugar deoxyribose, and a phosphate group.
The nucleotides are held together by a sugar-phosphate backbone, which consists of a deoxyribose
from one base covalently bonded to the phosphate group of the following base in a chain. The two
polynucleotide chains run anti-parallel to one another and are held together by hydrogen bonds, this
Figure 1. (a) cis structure, (b) trans structure and (c) superposition of both structures. (Wang et al, 2008)
hydrogen bonding follows the base pairing rules (A to T and C to G) which then forms the double-
stranded DNA (Watson and Crick, 1953).
The interaction between DNA and DNA helicase results in the unwinding of dsDNA which is
important in the replication and preservation of DNA in cellular organisms (Rasnik et al, 2002).
The DNA helicase-DNA interaction plays important roles in almost all parts of nucleic acid
metabolism. This includes; DNA replication, DNA repair, recombination, and transcription, all of
these processes rely on this interaction as some unwinding of dsDNA is needed to complete that
process (Rasnik et al, 2002).
The interaction of DNA helicase and DNA in prokaryotic DNA replication is triggered by; the protein
DnaA which recognises the origin of replication, and binds to a 9 base-pair sequence in the origin of
replication (Leonard and Grimwade, 2010). A pre-replication complex is formed once DnaA binds to
5 of 9 bp long sequences and 4 non consensus sequences within the origin of replication (Bryant and
Aves 2011). Once this pre-replication complex has been formed, it is activated by the hydrolysis of
ATP by DnaA resulting in the dsDNA unwinding at the origin of replication (Baker and Wickner,
1992). This denatured region of DNA is then available for DnaC helicase to load DnaB onto the two
separated DNA strands which travel in opposite directions, creating two potential replication forks
(Kobori and Kornberg, 1982). All proteins that are associated with the replication fork are linked with
DnaB. The energy required for the interaction of DnaC and DnaB is created from ATP hydrolysis
(Kobori and Kornberg, 1982; Fass et al, 1999). Once DNA helicase is active on the DNA strand, it
moves along the strand, breaking the hydrogen bonds between the dsDNA which also uses ATP to
provide energy for the interaction. The replication fork produced from the unwinding of DNA by
DNA helicase is held open by single-strand binding proteins so the DNA strands can be replicated
(Fass et al, 1999).
The interaction between DNA helicase and DNA occurs in the nucleus of a cell when DNA is
replicating, being repaired, recombined, or transcribed (Rasnik et al, 2002).
The incorrect function of DNA helicase results in many disorders, eg. XPD (Xeroderma pigmentosum
factor D) is a 5-3 ATP-dependant helicase that is an essential component of transcription factor II H
(TFIIH), this complex is involved in nucleotide excision repair by unwinding DNA (Fan et al, 2008).
XPD helicase point mutations have been found to be associated with three distinct phenotypes;
cancer-prone xeroderma pigmentosum (XP) or aging disorders Cockayne syndrome (CS) and
trichothiodystrophy (TTD). Mutations in various locations of the protein interactions, alteration of
the fluidity of XPD and alterations of the site of ATP or DNA binding site results in an unstable
complex and causes the above disorders (Fan et al, 2008).