Molecular Genetics 1.2

Block 1 - Lecture 2

What phase of the cell cycle does replication take place?

Synthesis phase (S phase)

How many potential origins of replication are there?

30-50,000 but only a subset are activated per S-phase

What happens in Pre-RC assembly in G1 phase?

Origin Recognition Complex (6-submit ATPase complex) binds DNA
Cdc6 & Cdt1 recruit MCM2-7 double hexamer 
DNA sequence
No strict consensus sequences 
Enrichment of OGREs (Origin G-rich Repeated Elements) 
Formation of G4 DNA - can act as recruit platforms for origin-binding proteins but as a barrier to fork progression if required
Helices such as PIF1 and BLM involved in unwinding G4s during replication

Origins are fired during S-Phase entry, what does this cause?

Origins fire once, then are inactivated for the rest of S-Phase

Activation of Kinases:
Cyclin Dependent Kinase 2/Cyclin E
Dbf4-dependent Cdc7 kinase (DDK)
phosphorylate initiation factors
minichromosome maintenance protein complex (MCM)

CMG complex encircles the leading strand template
ATP hydrolysis drives unwinding
Initiation factors (TopBP1, Treslin, RecQL4) stabilise
CMG assembly

What are replication forks and bubbles?

Replication forks form at the point of synthesis initiation - a Replication Origin

Two DNA strands are generated via co-ordination of Leading Strand and Lagging
Strand synthesis

Leading strand DNA synthesis is continuous in a 5ʹ – 3ʹ direction producing
as a single, continuous molecule, until the
replication fork terminates.

Lagging strand DNA synthesis is discontinuous in a 3ʹ – 5ʹ
direction producing as a series of “short” molecules Okazaki fragments, until the replication fork terminates

How is the replication fork built and name key molecules involved?

Unwinding: CMG separates dsDNA into ssDNA

Replication Protein A binds ssDNA stabilising via prevention of formation secondary structures

Polymerases:

Pol α-primase: RNA-DNA primer

Pol ε: continuous leading strand synthesis

Pol δ: discontinuous lagging strand (Okazaki fragments)

Processivity factors:

Proliferating cell nuclear antigen clamp keeps polymerase bound

Replication Factor C loads PCNA

Topoisomerases I/II relieve supercoiling ahead of fork

How is the lagging strand completed?

Lagging strand continuity is critical for genome integrity Okazaki fragments ~150 bp

Primer removal

RNase H1 digests RNA except last ribonucleotide

Flap Endonuclease 1 (FEN1) removes remaining flap structure

Gap filling

Pol δ synthesizes DNA

Nick sealing

DNA Ligase I joins fragments

Tell me about proof reading and error fixing in DNA transcription

Polymerase proofreading:

Pol δ & Pol ε have 3’→5’ exonuclease

domains

Mismatch Repair (MMR):

MutSα (MSH2/MSH6) detects mismatches and INDELs

MutLα (MLH1/PMS2) directs excision and resynthesis

Error rates:

Pol error: ~10⁻⁵ / bp

With proofreading: ~10⁻⁷ / bp

With MMR: ~10⁻¹⁰ / bp

Replicating telomeres

Lagging strand cannot fully replicate ends of DNA so Telomerase (TERT + TERC RNA template) extends 3’ overhang

Shelterin complex (TRF1, TRF2, POT1, TPP1, TIN2,

RAP1):
Protects ends from DNA damage response
Prevents inappropriate repair/recombination
Telomere shortening results in cellular senescence, aging
Telomerase reactivation in observed cancer immortality

Alternative Lengthening of Telomere (ALT) pathway in telomerase deficiency

True or false? Transcription & RNA polymerases require primer molecules to begin polymerisation?

FALSE! They do not require primers

In which direction do RNA polymerases synthesise transcripts?

Synthesised in a 5’ to 3’ direction, therefore creating an RNA equivalent of the gene DNA sequence. Transcript sequences are always written in a 5’ to 3’ direction, from left to right. 

What are the key molecules in transcription?

RNA Pol I: rRNA

RNA Pol II: mRNA, lncRNA, snRNA

RNA Pol III: tRNA, rRNA and small RNAs

RNA Pol 2

Catalyses the formation of phosphodiester bonds in the sugar phosphate backbone of RNA

DNA-directed synthesis of mRNA during transcription

550kDA complex of 12 subunits

RNA polymerase cannot enter any part of transcription alone

What is the difference between gene and gene specific transcription factors?

General transcription factors (GTFs)
Usually specific for each class of RNA polymerase
May directly or indirectly interact with the RNA polymerase, at a promoter, to initiate formation of a Pre-Initiation Complex (PIC) and facilitate the transcription process

Gene-specific transcription factors Respond to cell signals and determine which genes are transcribed. Can act to activate or repress transcription

What are promotor elements and give examples?

Sequence of DNA to which proteins bind to initiate transcription downstream

TATA box (-25 to -30) - TATA(A/T)A(A/T)

Binds TATA Binding Protein
Positions RNA Pol II at transcription start site
Initiation focused at a single TSS

Initiator - PyPyAN(T/A)PyPy (A = TSS)
Overlaps with Transcription Start Site
Recognised by TAF/TAF2
Supports multiple dispersed start sites

Downstream Promotor Elements (+28 - +32)
Functions in TATA-less promotors
Recognised by TAF6/TAF9