Molecular Motors: Helicase

Question 1

What are Molecular Motors?

Answer 1

• Molecular motors are biological nano-machines involved in key cellular processes
• 3-dimensional structure is critical to their function
• Utilise chemical energy from ATP/GTP to perform mechanical work
• Have complex structure and employ ‘Switch mechanism’ via changing their conformation during their function

Question 2

What are the different types of Molecular motors?

Answer 2

• Cytoskeletal motors: Mysosin, Kinesin, Dynein
• Rotary motors: ATP-synthase, Flagellum motors
• Nucleic Acid Motors: Helicases

Question 3

Give an introduction of Helicases ?

Answer 3

• Nuclear proteins and first discovered in E. coli in 1976
• Ubiquitous and abundant
• Unwind ssDNA or dsDNA helix and removes secondary structures in RNA
• Change conformation by hydrolysing ATP to translocate along the DNA or RNA chain
• Move or translocate along the DNA or RNA
  5’ to 3’ or 3’ to 5’
• Function in a variety of oligomeric forms – dimers and hexamers are most common
• Helicases are essential and implicated in genetic disorders

Question 4

Human genetic diseases associated with helicases. Give an example ?

Answer 4

Mutant BLM Helicase causes disease

• autosomal recessive
• growth retardation
• “butterfly” rash due to sun exposure
• compromised immune system
• elevated risk of cancer

Question 5

What do all helices share?

Answer 5

• They share a RecA-like fold

- Conserved Helicase motifs

Question 6

What do all helicases contain?

Answer 6

All Helicases contain an ATP binding site and a separate nucleic acid binding site

Question 7

How many superfamilies does helices have ?

Answer 7

Six superfamilies based on primary structure

Question 8

Explain function of Helicases: DNA replication/DNA repair?

Answer 8

Each DNA strand needs to be separated to act as template for a new complimentary strand

Question 9

Explain function of Helicases: Transcription?

Answer 9

DNA strands have to be separated so that they can be used as a template to produce an mRNA molecule

Question 10

Explain function of Helicases: RNA secondary structures and DNA-RNA hybrids?

Answer 10

These structures arise during transcription. For further use of the mRNA as a template for translation, it is critical that the RNA is separated from DNA and also that the nascent RNA is devoid of any secondary structures to avoid stalling of the ribosome

Question 11

Classification of Helicases:

DNA helicases ?

Answer 11

• Unwind duplex DNA to create single stranded DNA intermediates that are required for DNA replication, transcription, repair and recombination
• Translocate branched DNA structures eg. D-loops, Holliday junctions

Question 12

Classification of Helicases:

RNA helicases ?

Answer 12

Destabilise RNA secondary structures, promote translation, RNA splicing, transport and degradation

Question 13

Classification of Helicases:

DNA/RNA helicases ?

Answer 13

Unwind DNA/RNA hybrid structures, regulate DNA replication initiation, transcription and termination

Question 14

What are some Helicase Activity Parameters ?

Answer 14

1. Rate: Number of base pairs unwound per unit time
2. Processivity: Average number of base pairs unwound by each helicase molecule per encounter with DNA substrate
3. Step-size: Average number of base pairs unwound per ATP hydrolysis cycle

Question 15

What are the two functions of Bonafide Helicases ?

Answer 15

1. Need single-stranded nucleic acid region to bind and to initiate their action of strand separation
2. Base pair separation occurs at the junction of single-stranded and duplex regions and it translocate ssDNA or RNA

Question 16

Function of Translocase ?

Answer 16

1. Binds double stranded DNA and moves along/pulls DNA

Question 17

Role of Helicase activity ?

Answer 17

1. DNA/RNA unwinding (ie helicase) activity
2. DNA/ RNA remodelling activity
3. Nucleoprotein complex remodelling activity
4. Protein motion on DNA duplex

Question 18

What are the different structures of Helicases ?

Answer 18

1. Non-hexameric Helicase: PcrA

2. 2. Hexameric Helicase: HPV E1

Question 19

Explain PcrA Helicase ?

Answer 19

• Plasmid copy number reduction A
• Helicase in gram-positive bacteria
• Member of SF1 family. Type A (3’ to 5’ activity)
• Functional role in DNA repair and rolling circle replication mechanism
• ssDNA translocation motor

Question 20

What is the Inchworm mechanism ?

Answer 20

Mechanism of dsDNA strand separation and translocation by PcrA

Question 21

What does PcrA helicase have a homologous structure to ?

Answer 21

PcrA helicase has a homologous structure to Bloom (BLM) helicase which causes human diseases

Question 22

What does understanding PcrA helices activity shed light on ?

Answer 22

How mutant BLM helices works

Question 23

Explain Hexameric Helicases ?

Answer 23

• Superfamilies 3 to 6
• ATP binding site at the interface between monomers
• Toroidal structure confers high processivity
• Some helicases self-assemble around the duplex while some require additional factors to dock onto the double stranded DNA substrate
• ATP binding site exhibit strong negative cooperativity between them. Up to 4 sites per complex are occupied by ATP

Question 24

Give brief background of HPV E1 helices structure ?

Answer 24

• Helicase of human Papillomavirus
• Viral DNA replication initiation
• Cause cancer in humans
• Forefront of research

Question 25

Explain structure of HPV E1 helices structure ?

Answer 25

• Hexamer of HPV E1
• ssDNA bound in central channel
• ATP binding pocket status changes sequentially around ring

Question 26

Explain the Histidine loop when ATP is bound and when ATP is empty ?

Answer 26

• When ATP is bound, the Histidine loop is at highest position
• When empty, the loop is at lowest position