DNA Replication

3/20/24


Rules of Replication

Requirements:

  1. Speed: Very fast

    1. 1000 BP per secon in E.coli- one bubble in a circle

    2. 50 BP per second per replication fork in humans

    3. Multiple bubbles= 1 chromsomes in a few hours

  2. Fidelity: Few mistakes

    1. the mutatin rate (SNP) in human is between 1e^-8 to 2.5e^-8 per base per generation

      1. this means roughly ~1 mistake in 100 million base pairs

  3. Organization:

    1. DNA should not be broken or wound into knots etc. If this happens it should be repaired!

Rules of Replication

  1. Strands are complementary

  2. the two strands are separate

  3. Both strands serve as a template

  4. New DNA is synthesized 5’ to 3’

  5. DNA is synthesized by DNA polymerase

1) Strands are Complimentary

  • Antiparallel and complimentary

  • DNA sequence: 5’ TGTA

    • 3’ TACA (read 5’ -3’ so reversed)

2) The Two strands are separate

  • The Y shaped junctions on either side where the two strands are separating are called replication forks.

  • The structure where the two strands are pulled apart is called a bubble, in this case a replication bubble.

3) Both strands serve as a template

  • The semi conservative model of DNA replication. After synthesuis of the new strands, each original strand forms a DNA duplex with the newlty synthesized strand that is complementary in sequence

4) New DNA is synthesized 5’-3’ by DNA polymerase

  • A DNA polymerase is not one protein, it is a huge protein complex with multiple proteins (a holoenzyme)

  • DNA polymerase active site

  • DNA polymerase: Synthesizes new strands of DNA from an ssDNA template in the 5’ to 3’ direction by catalyzing phosphodiester bonds


Replication in Bacteria

  • Replication starts in the origun of replication

  • Unwinding the DNA strands creates a replication bubble

  • For each replication bubble there are two replication forks which open continuously as DNA is synthesized in opposite directions

Replication Requires Proteins

  • Prokaryotic DNA replication

  • Polymerase is shortened to pol- there are multiple types of DNA polymerase

  • SSB stands for single stranded binding protein

  • Topoisomerase is also called Gyrase

  • DNA binds to the origin and initiates replications

  • An Ori is a sequence of DNA- In real DNA sequence there is a common pattern in a binding site (sometimes called a box) but the specific sequence has some variation

    • DnaA proteins bind to specific sequences in the origin called DnaA boxes

DNA Sequences Where Proteins Bind

Consensus Sequence- the m ost commonly observed base at eavh position in a functional sequence a specific sequence exactly

Motif- a functional sequence represented by a pattern of the miost likely bases at each position


Steps in Bacteria DNA Replication

Step 1) Initiation

  • DnaA proteins binds to DnaA boxes

  • DnaA complex bend DNA initiating replication. by creating an initail opening

  • DnaC Protein- Delivery factor

  • DnaB Protein- the helicase


  1. Helicase breaks hydrogen bonds, Topoisomerase relaxes supercoiling and prevents DNA tangling

  2. Replication is initiated by opening the DNA at the origin of replication (ori).

  3. Single Stranded Binding Protein (SSB) prevents reannealing

Step 2) Beginning DNA synthesis with RNA

  • A Primer is required for Synthesis added by primase- this proves the free 3’ OH for DNA polymerase

  • Bases are added to the 3’ end building the DNA in the direction of 5’ to 3’ by DNA polymerase II

  • DNA Polymerase III Synthesizes daughter strand. Leading strand SSB has been deleted for clarity

  • DNA polymerase III elongates the leading strand continuously and the lagging strand discontinuously.

Error Correction

  • DNA polymerase III:

    • 3’ to 5’ exocnulease (think erasing) activity ti correct errors

    • 5’to 3’ for normal activity for synthesis

Leading and Lagging Strand

  • Leading Strand: 5 to 3 into each fork

  • Lagging strand synthesis away from each fork


Prokaryotic Elongation

  • DNA polymerase I removes and replaces the RNA primer bases

  • DNA ligase joins Okazaki fragments

  • Finally nicks in the strand are sealed by the enzyme ligate

Stopping the Process

  • In E.Coli Tus (terminus utilization substance) protein binds to termination sequences and stalls the replication fork terminating the replication process

  • Interlocking rings of DNA are separated by topoisomerase

    • Product is 2 circular chromosomes that are half old template dna and half newly synthesized DNA




Steps in Eukaryotic DNA Replication

  • We have more chromosomes and ours are linear

  • A replicon is a region of replication

    • Bacteria have 1 replication origin whereas eukaryotes have many origins and multiple replicons during synthesis

  • In eukaryotes, replication is licensed and replication is only allowed once per origin per round of DNA synthesis

  • regular synthesis will leave gaps at the 5’ ends when the primer is removed

  • DNA polymerase cant add bases to a 5’ end, there is no free 3’ OH group

  • A specialized polymerase called telomerase is brought in to extend ends and keep DNA at telomeres from beginning shorter each round of replication

    • Translocation of telomerase

    • Elongation of DNA

  • The ends (telomaeres) then consist of disctinct repetitive DNA and form a protective structure called a T loop

    • Telomaere completion (by DNA polymerse)

  • If the T loops break down and ther is no telomerase, programmed cell death (apoptosis) occurs, this determines a cell’s life span and how many times it can go through the cell cycle before it dies