detail description about different models of DNA helicase. basically 2 types of mechanism involved in the helicase function and it has various models. this information is refer from research paper and some review articles.
3. INTRODUCTION
• Double stranded(ds) DNA is the most stable form of most the
DNA in vivo.
• This ds DNA must be unwound to provide the single stranded
(ss) DNA intermediates require for the DNA metabolism.
• The unwinding of ds DNA during DNA replication,
recombination and repair is catalysed by a class of enzyme
termed as DNA helicase.
4. HELICASE
• Helicase are enzymes which are motor protein
that move directionally along a nucleic acid and
separating two nucleic acid strand using energy
driven from ATP hydrolysis
5. TYPES OF HELICASE
• According to their substrate
1)DNA Helicase
unwind duplex DNA &form ss DNA
Require for DNA replication, repair &recombination
2) RNA Helicase
destabilized secondary structure of RNA
promote RNA splicing, editing, transport °radation
6. MECHANISM
• Two Types :
1 Translocation mechanism
• Helicase catalysis repeated cycles of base pair separation coupled with
translocation.
• Some helicase have ability to translocate uncoupled with base pair
separation
• This mechanism involve NTPase coupled with nucleic acid affinity changes &
conformational changes.
2 Base pair separation mechanism
• This mechanism occurs at the junction of single stranded & duplex region.
• Helicase unwind long stretch of nucleic acid coupled with translocation.
8. • For monomeric helicase, one helicase site is tightly bind with
nucleic acid where as other is weakly associated with nucleic
acid
• Weak site dissociate from nucleic acid and in power stoke
motion moves away from tight site to bind at position ahead
• Then the original tight site becomes weak
• One cycle is completed in six conformation changes
9. Active rolling model
• This model proposed for dimeric helicase used for DNA
unwinding
• Each subunit either bind with single stranded DNA or
duplex DNA
• Have a longer step size (4 – 5 base pair)
10. Brownian motor model
• Involve Brownian motion and power stroke which based on two
conformation state of helicase
• Tight state :Helicase nucleic acid energy profile is deep and sawtooth shaped
Helicase movement is not possible
• Weak state : Helicase nucleic acid energy profile is shallow and symmetric
Helicase can move in either direction or completely dissociate
(low processivity)
• When helicase resume tight state it makes step forward (power stroke)
• Some molecules shift in forward direction , moves ahead while some are
shift in opposite direction, return to their original position.
• Repetition of these step leads to net forward movement of helicase along
nucleic acid
11.
12. Brownian model
1. One nucleic acid binding
site
2. Two conformational
changes
3. Both Power stroke
motion and Brownian
motion
Stepping model
1. Two nucleic acid
binding site
2. Six conformational
changes
3. Power stroke
motion
Difference
14. • MCM Complexes load onto DNA at the origin of replication and
moves away from the origin.
• For this step, its important that all MCM hexamer face the same
direction
• Their orientation is specified by ORC in association with cdc6 and
Cdt1,which are require for helicase loading
• Dispersed MCM are anchored to immobile nuclear structure
• Now only DNA will be able to rotate
• Rotation of DNA in opposite direction from the two side of the
replication bubble unwind the DNA at the replication fork
15.
16. ds DNA PUMP MODEL
• “Rabbit ear” electron micrograph & high
resolution of X ray crystal structure of
SV40 Tag favour the ds DNA pump
model
• In SV40 DNA replication, host cellular
replication machinery utilized except for
MCM helicase.
• SV40 large antigen have helicase
function.
• SV40 LT hexameric helicase recognise
and bound to origin of ds DNA.
• SV40 LT load on to origin as head to
head double hexamer
17. • SV40 LT have 3 domain: 1) Dna J homology domain
2)Origin binding domain
3)helicase domain
• Helicase domain consist AAA+ domain (ATPase associated with cellular
activity) , which catalysis ATP hydrolysis
• Origin of SV40 DNA is 64base pair long ,which have 32bp long AT half and
32bp long early palindromic half (each contain 2 GAGGC penta nucleotide
sequence and AT rich seq)
FEATURES OF SV40 LARGE T ANTIGEN
18. • The origin of ds DNA inside the narrow region of AAA+
domain of helicase shows a partial melting around the AT
rich region due to compression of two sugar- phosphate
backbone
• Forcing the Watson crick base pairing within the duplex to
flip outside of the complex
19. Ploughshare model
• It was suggested that the MCM complex translocate along
duplex DNA and strand separation is achieved by protein pin
that separate two strands.
• This mechanism require initial melting of DNA to allow the
binding of protein pin between strands
• This protein may be provided either by MCM helicase (after
DNA melting and enzyme activation) or by newly loaded
protein
20.
21. Steric exclusion model
• This model were proposed after illumination of
Salfolobus solfataricus MCM helicase crystal
structure
• MCM hexamer contain a central channel that
encircle DNA , N- terminal tier and C terminal AAA+
ATPase tier
• N- terminal tier acts to increase the processivity of
the helicase by modulating the interaction with DNA.
• C- terminal AAA+ domain that involve in ATP
hydrolysis
• Beta hairpin of central channel catalyse the
unwinding activity
• One strand pass through central channel and other is
displaced away from exterior surface of helicase
