Mukul Dev Yadav 1

INTRODUCTION

STRUCTURE OF HELICASE

CLASSIFICATION OF HELICASE

MECHANISM OF HELICASE ACTION

HELICASE AND HUMAN DISEASE

CONCLUSION

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The family of nucleic acid strand separation enzyme known as Helicase.

This enzyme found in all organisms and participate in wide variety of cellular

processes.

The number of helicase expressed in higher organisms is strikingly high, with

approximately 1% of the genes in many eukaryotic genomes apparently encoding

RNA or DNA helicase.

Helicases are involved in virtually all aspects of nucleic acid metabolism,

including replication, repair, recombination, transcription, chromosome

segregation, and telomere maintenance .

Traditionally, helicases are known to unwind double-stranded DNA or RNA in an

ATP-dependent manner.

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All helicases share a RecA fold.

Each helicase molecule contains a single NTP binding site and a distinct

polynucleotide binding site.

These sites are allosterically linked, since the NTPase activity modulates nucleic

acid binding affinity, and vice-versa.

Common Features of Helicase Structure

Sub-domains 1A and 2A contain RecA-like folds. Residues from conserved

helicase motifs line the interface of 1A and 2A and bind NTP.

Single-stranded nuleic acid binds in a groove that is formed by 1A and 2A sub-

domains.

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Conserved helicase motifs and NTP binding site are at subunit interface.

A critical arginine residue from a neighboring subunit is within hydrogen-

bonding distance of the gamma phosphate of NTP bound at the interface, and is

implicated in transducing conformational changes between subunits of the

hexamer.

The central channel of the ring is large enough to accommodate a single strand of

DNA or RNA.

Rings appear to adopt multiple asymmetric conformations in response to ligand

binding.

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Fig 1. Crystal structure of bacteriophage T7 gp4 helicase domain.

A) shows the six helicase subunits in a ring with a C2 symmetry and four ADPNP (dark blue) bound

at the subunit interface. Symmetrical subunits are shown in same colour .

B) Helicase conserved motif of T7& gp4 protein. Gray area represents the helicase domain shown in

the crystal structure.

A) Stereo view from inside the ring of subunits A and B complexed to ADPNP (dark blue) and Mg

(green space filing). The helicase conserved motifs are shown in the same colour as in (B). The

subunits are coloured the same as in (A).

Hexamer Helicase Structures

On the basis of Directionality

Type A- These type helicases unwind the nucleic acid strand in 3’-5’direction.

These helicases requires a 3’tail on the substrate duplex.

Type B- These type of helicases unwind the nucleic acid strand in 5’-3’direction.

These helicases require a 5’tail on the substrate duplex.

Fig 2. Schematic of an unwinding reaction catalyzed by 3’-5’Helicase.

Some helicases have ability to initiate unwinding from a blunt ended duplex.

E.g. – Rec BCD system in E.coli

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DNA Helicases

It translocate on DNA lattices.

Unwind duplex DNA to form ssDNA intermediates required for DNA replication,

recombination, repair.

Process, translocate branched DNA structures-- Holliday junctions, D-loops, etc.

RNA Helicases

It translocate on RNA lattices.

Destabilize RNA secondary structure; promote ribosome assembly, translation,

RNA splicing, editing, transport, & degradation.

DNA/RNA Helicases

Unwind RNA/DNA hybrids; transcription termination, regulate DNA replication

initiation, etc.

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On the basis of sequence level helicases divided into Six main group i.e., super

families one to six. This grouping based on their shared sequence motif.

Super Family-1 (SF-1)

Super family -1 can be further divided in to two classes

SF-1A helicases- These helicases have 3’-5’ polarity.

SF-1B helicases- These helicases have 5’-3’ polarity.

But maximum member of SF-1 have 3’-5’ directionality.

It contain seven so called helicase motif I, Ia, II, III, IV,V,VI.

Most known helicases of

SF-1A- Rep & Uvr D in gram negative bacteria.

Pcr A in gram positive bacteria.

SF-1B- Rec D & Dda helicase.

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This is the largest group of helicase.

It is characterised by the presence of nine conserved motifs like Q, I, Ia, Ib, II, III, IV, V,

VI.

They possess mainly 3’-5’ directionality i.e. type A helicases. But there are some

exceptions like XPD family which is has a polarity of 5’-3’ i.e. type B helicases.

Motif I & II are same as SF-1, but other motif differ in conserved sequences .

Motif I, II, Q, VI all needed for ATP binding & hydrolysis. Motif Ia, Ib, III, IV, V may be

involved in intra molecular rearrangement and RNA interactions.

E.g. Dead box RNA Helicases. XPD (Xeroderma Pigmentosum factor-D) family.

Super Family-3(SF-3)

SF-3 consist of helicase encoded mainly by small DNA viruses and large nucleocytoplasmic DNA

viruses. These are mainly of type A helicases i.e. have 3’-5’ polarity.

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SF-3 family contain three conserved motifs as motif I, II, III.

E.g.- Papilloma virus have E-1 helicase.

Super Family-4(SF-4)

All SF-4 family helicase have type-B i.e. have 5’-3’ polarity.

These enzyme present in ring structure, mainly present in hexameric ring

structure.

The most studied SF-4 helicases is gp4 (gene 4 helicase-primase) from

bacteriophage T7.

gp4 helicase is member of ring-shaped family of helicases.

Super Family-5(SF-5)

SF-5 is a small family have enzyme such as bacterial transcription factor Rho.

Rho is an essential transcription protein in prokaryotes.

It function by wrapping nucleic acid around a single cleft extending around the

entire hexamer.

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In recent works SF-6 also called AAA+ protein (ATPase associated with diverse

cellular activities).

It has some enzyme such as RUB branch migration enzyme and MCM proteins

( minichromosome protein complex).

MCM is a eukaryotic DNA helicase complex required for the process DNA

replication, specifically formation and elongation of replication fork. It is

hexamer of six related polypeptide that forms a ring structure.

MCM is also a component of the pre replication complex that form on eukaryotic

ori.

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Helicases couple the chemical energy of NTP binding and hydrolysis to separate

the complementary strands of double-stranded nucleic acids, remove nucleic acid

associated proteins, or catalyze homologous DNA recombination.

The helicase function is required for efficient and accurate replication, repair, and

recombination of the genome. Similarly, helicase functions facilitate RNA

metabolic processes such as transcription, ribosome biogenesis, translation, RNA

splicing, RNA editing, RNA transport, and RNA degradation.

Here, we focus on the strategies that helicases use to translocate and catalyze

strand separation coupled to NTP binding and hydrolysis.

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Active State of Helicases

Monomers of ring-shaped helicases are not active in catalyzing NTPase or

unwinding reaction, and hexamer formation is essential.

The ring-shaped structure is stabilized by the binding of NTP, a metal ion, or

both, and by the nucleic acid substrate .

The enclosure of the nucleic acid by the protein subunits decreases the

probability of helicase falling off, thus increasing the ability of the helicase to

stay on track.

Another advantage of this arrangement is the coupling of NTPase cycles

between the hexameric subunits that can increase the efficiency of the

NTPase cycles in promoting translocation.

Oligomerization is an important strategy for non-ring-shaped SF1 and SF2

helicases as well.

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Some helicases utilize a structural interaction whereas others rely on a functional

interaction. Structural interaction results in the formation of a homodimer or

heterodimer that converts the helicase into a more effective enzyme.

Many helicases show functional cooperativity and enhanced processivity when

multiple molecules of helicases are loaded on the tracking strand .

Yet their activity is enhanced when multiple helicases are loaded on the tracking

strand, which is attributed either to prevention of backward helicase slips or

simply the availability of additional helicase molecules when one falls of the

track.

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Fig 3. Functional oligomeric states of helicases on nucleic acids.

a, ring-shaped hexamer.

b, helicase monomer.

c, helicase dimer.

d, heterodimer.

e, higher oligomer .

Most helicases need a single-stranded nucleic acid region to bind and to initiate

their action of strand separation. Once loaded on the strand, they show a

directional bias and translocate either 5′–3′ or 3′–5′.

Ring-shaped helicases require Y-shaped nucleic acid structures with a loading

strand and a noncomplementary strand of an optimum length to initiate

unwinding.

Helicases show different degrees of tolerance to changes in the chemical nature

of the loading strand while translocating. Some are sensitive to breaks to a basic

sites or to electrostatic disruptions.

While unwinding , certain helicases show no sensitivity to the nature of the

displaced strand. On the other hand, the nature of the displaced strand appears to

influence the activity of some helicase.

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Fig 4. Modes of interacting with the nucleic acid substrate .

a, helicase interacts with one of the single strands of the nucleic acid near the

unwinding junction.

b, helicase interacts with one of the single strands and the duplex region near the

unwinding junction.

c, helicase interacts with both strands of the nucleic acid.

d, helicase interacts with the duplex region.

e, helicase interacts with both the duplex and the two strands of the nucleic acid.

All of the mechanisms involve NTPase coupled nucleic acid affinity changes and

a conformational change (power stroke or ratchet) to explain biased movement

that results in base pair separation or translocation.

The differences in the proposed mechanisms reflect the diverse biochemical

properties including the oligomeric state of the helicase, its mode of binding the

nucleic acid at the unwinding junction, and the effect of the NTP ligation state on

nucleic acid binding properties.

Stepping Mechanisms

In the stepping models, the helicase is always bound to the nucleic acid via two

nucleic acid binding site.

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In an inchworm type stepping model for a monomeric helicase, a cycle of

nucleic acid binding, release, and translocation events begins with one

helicase site bound tightly to the nucleic acid and the second helicase site

bound weakly to the nucleic acid .

The weak site dissociates from the nucleic acid and in a power stroke motion

moves away from the tight site to bind at a position ahead.

After the weak site has moved and made tight interactions ahead, the original

tight site becomes weak, and as it dissociates from the nucleic acid, in a

power stroke motion it moves forward to get close in distance to the site

ahead.

One cycle in an inchworm stepping mechanism is completed in six

conformational changes.

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An alternative stepping mechanism (rolling model) for a dimeric helicase has

been proposed for DNA unwinding .

In this model, each of the two subunits of the helicase alternate their binding to

single-stranded and duplex DNA as they change their NTP ligation states.

In contrast to the inchworm model, where the subunits maintain their relative

positioning along the DNA, the subunits in the rolling model take turn in being

the trailing or the leading subunit.

Brownian Motor Mechanism

This model invokes Brownian motion and power stroke, and it is based on two

conformational states of the helicase.

Structural and biochemical studies have identified two distinct conformational

states of helicase with weak and tight nucleic acid binding modes resulting from

the different NTP ligation states.

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To translocate the helicase needs to loosen its interactions with the nucleic acid,

and this happens when the helicase changes its NTP ligation state.

In the weak state, the helicase-nucleic acid energy profile is shallow and

symmetric, and the helicase can move in either direction (Brownian motion) or

completely dissociate from the nucleic acid.

The short lifetime of the weak state keeps the helicase close to the starting

position. When the helicase resumes the tight state, it makes a step forward

(power stroke).

Those molecules that have fluctuated in the forward direction move ahead and

those that have fluctuated in the opposite direction return to the original position.

Repetition of these steps leads to net forward movement of the helicase along the

nucleic acid.

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Fig 5. Proposed mechanisms of translocation(A, stepping inchworm mechanism. A helicase monomer with a

tight (closed hand) and a weak (open hand) nucleic acid binding site is shown to undergo steps of helicase

movement (power stroke) and nucleic acid affinity changes (tight to weak transitions).

B, Brownian motor mechanism. On the right, the helicase is shown to undergo nucleic acid affinity changes

(tight to weak). In the weak state (2), the helicase fluctuates in either direction. Upon resuming the tight state

(3), some helicase molecules move forward (3) and some return to their original position (1). On the left, the free

energy of the helicase-nucleic acid complex is shown along the nucleic acid length. In the tight state (1), the

helicase is trapped in a deep energy well unable to move. In the weak state (2), thermal fluctuations allow the

helicase to fluctuate in either direction or to completely dissociate from the nucleic acid (4). Upon resuming the

tight state, the deep energy proነle is restored and some helicases move forward (3).

Base pair separation occurs at the junction of single-stranded and duplex regions.

Helicases unwind long stretches of duplex nucleic acids by coupling base pair

separation to translocation.

Depending on how the base pairs are separated, the base pair separation

mechanisms are classified as active or passive .

If the helicase needs to move and bind more than one base at a time, it would

employ some type of an active mechanism to bring about strand separation in an

efficient manner.

In a passive mechanism, the helicase waits for the base pairs to open

spontaneously by thermal fluctuations before it moves and binds the newly

opened bases. Because the terminal base pair at the junction opens and closes at a

very fast rate , this type of a mechanism is attractive for helicases that can move

and occupy one base at a time.

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Fig 6. Proposed mechanisms of strand separation.

A, helicase separates the base pairs at the junction by translocating along one strand of the nucleic acid while

displacing the other using strand exclusion, wire stripper, or a wedge mechanism..

B, helicase destabilizes the duplex region by direct interactions (helix destabilizing mechanism).

C–F illustrate proposed mechanisms for ring-shaped helicases catalyzing bidirectional replication fork

movement. C, helicase rings encircle one of the DNA strands and move in opposite directions to unwind DNA by

strand exclusion mechanism. D, helicase rings encircle duplex DNA and unwind DNA by a

torsional mechanism. E, helicase rings encircle and pump duplex DNA and separate the strands by threading

them through side channels. F, helicase rings encircle and pump duplex DNA and separate the strands by a

Ploughshare mechanism (blue triangle).

Translocation and base pair separation activities of helicases are driven by NTP

binding and hydrolysis.

During each NTPase cycle, the helicase goes through defined NTP ligation states

including empty, NTP, NDP*Pi, and NDP. One or more of the NTP ligation states

causes changes in the affinity of the helicase for the nucleic acid and brings about

a power stroke that leads to translocation and/or strand separation.

Power stroke and nucleic acid affinity modulation steps are energetic events;

therefore, only those changes in the NTPase cycle that are associated with an

observable energy change are likely to drive these events at the nucleic acid

binding site.

Ring-shaped helicases potentially can bind and hydrolyze six NTPs, and many

show co-operativity in NTP binding and hydrolysis.

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Helicases are required for the efficient catalysis of most DNA and RNA

metabolic processes where they perform diverse functions.

All helicases share a RecA fold. Each helicase molecule contains a single NTP

binding site and a distinct polynucleotide binding site.

Most helicases need a single-stranded nucleic acid region to bind and to initiate

their action of strand separation.

Base pair separation occurs at the junction of single-stranded and duplex regions.

The basic activity of helicases is to couple NTP binding and hydrolysis to

conformational changes that bring about separation of base pairs or translocation

along nucleic acid.

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