DNA Replication

DNA replication

Process of copying DNA

Nucleotide

Made up of/building blocks of DNA:

Phosphate group

Deoxyribose sugar

Nitrogenous Base

Pyrimidines

Cytosine

Thymine

Purines

Adenine

Guanine

DNA Structure

Double Helix

Antiparallel (3’-5’ and 5’-3’)

Sugar Phosphate Backbone

Complementary Base Pairing (hydrogen bonds)

-A and T have 2 H-bonds

-C and G have 3 H-bonds (stronger than A and T)

Major groove and Minor groove

Double Stranded

Each new DNA molecule has:

1 old strand and 1 new strand

Having 1 new and 1 old strand is called:

Semi-conservative Replication

needs to be antiparallel

Meselson-Stahl Experiment

What they tested:

Conservative vs semi-conservative replication

They grew bacteria on heavy nitrogen N-15 and light nitrogen N-14

These would produce a band at the bottom or the top no in-between

Results showed a band in the middle demonstrating…

What they found:

DNA is semi-conservative!!!!

DNA polymerase

An enzyme that builds new DNA strands by adding nucleotides to a growing chain during DNA replication

Synthesizes DNA from the 5’-3’ direction

-Can only add on to already present strands, CANNOT start from scratch, needs a primer

-Needs a single-stranded template (daughter strand) so it knows which base to add

-Has proofreading ability (3’-5’ exonuclease activity) “from the end-chewing”

DNA polymerase III

Does bulk of DNA replication

Main synthesis

DNA pol I

Removes RNA primer and replaces it with DNA

Involved in replication on the lagging strand- has 5’-3’ exonuclease activity in addition to 3’-5’ exonuclease activity

Exonuclease

An enzyme that finds mistakes and allows for DNA pol to fix it

Where does replication start?

Ori C region

Ori C

Specific DNA sequence in bacteria where DNA replication begins

Where does replication end?

ter

stands for “termination” on the opposite side of Ori C

ter

Specific DNA region where replication ends in bacteria

Replication is what?

Bidirectional

Theta replication (progresses in both directions of the circle)

Two replication forks moving opposite ways to ultimately separate into 2 chromos

SSBP, Single-Stranded Binding Proteins

Proteins that bind to DNA strands to keep them from rejoining during replication

DNA B Helicase

An enzyme that unwinds the DNA double helix by breaking hydrogen bonds between the two strands during replication

Leading Strand

Continuous synthesis

Follows the replication fork

Only has primer laid down at Ori C

Lagging Strand

The strand is discontinuous and synthesized in small segments/pieces (Okazaki fragments) in the opposite direction of the replication fork

Okazaki fragments

Short pieces of DNA that are made on the lagging strand during DNA replication are later joined together

Lagging Strand Steps

1. Primase adds RNA primer

2. DNA Pol III extends DNA

3. DNA Pol I removes the RNA primer and replaces it with DNA

4. DNA Ligase seals the fragments together

RNA Primer

Short RNA sequence that provides a starting point for DNA polymerase to begin DNA synthesis

Primase

An enzyme that makes short RNA primers needed to start DNA replication

DNA Ligase

An enzyme that joins Okazaki fragments by sealing the sugar-phosphate backbone

GLUE!

DNA Gyrase

Relaxes supercoils ahead of replication fork

Topoisomerase II

An enzyme that cuts both DNA strands to relieve twisting and then reattaches them during replication

Replication Termination in E. Coli Sequence

1. Replication starts at Ori C

2. Forks move in opposite directions

3. Tus and Ter stop forks in termination region

4. DNA finished replicating

5. Topoisomerase II separates the linked circles

Ter sites

Specific DNA sequences = “stop signs”

Tus Proteins

Proteins that bind Ter sites