DNA replicationDNA replicationSections 8.3 -8.5Sections 8.3 -8.5

Biology 391Biology 391

DNA Synthesis• Who? - all living organisms• What? - making an exact duplicate of DNA• When? - during S phase of interphase• Where? – nucleus (or cytoplasm)• How? – via several enzymes

– semi-conservative– Denature, Anneal Primers, Extend

• Why? – So that we can grow (divide)

DNA Structure• Sugar-Phosphate

backbone• Nitrogenous Base

– C always pairs with G– A always pairs with T– Hydrogen bonds connect

• Antiparallel – 5’ phosphate– 3’ hydroxyl

Steps of DNA replication1. DENATURE

– Binding of enzymes to existing DNA• At replication origins• Replisome: DNA and associated bound proteins

– Double helix is unwound2. ANNEALING

– 4-15 nucleotides of RNA added to ssDNA3. EXTENSION

– Synthesis of a new matching strand for each existing strand

– Leading versus Lagging strands

Enzymes Involved• Helicase – separate initial DNA strands

– SSBP – prevent reannealing of DNA strands

• Primase – lay down initial RNA primers so DNA pol III can work

• DNA polymerase I – removes RNA primers (exonuclease) and replaces with DNA (CC#1)

• DNA polymerase III – main replication enzyme• DNA Ligase – “glue” together small DNA

fragments, seal backbone

DENATURING:starting DNA replication

• Replication Origins– A-T rich; easiest to break Why?– DNA is opened by initiator proteins and helicase– Different in Euks/Proks

Eukaryotes vs. Prokaryotes• Linear chromosomes• Multiple replication

origins• Synthesis occurs in

nucleus• Only works in one

direction• DNA pol adds ~100

bases per second

• Single circular chromosome

• One origin of replication• Synthesis occurs in

cytoplasm• Bidirectional• DNA pol adds ~1000

bases per second

ANNEALING:

• DNA Pol III needs a double stranded starting point, but replication requires the DNA to become single-stranded:– Solution: Put down short, temporary starter

sequences.– Primase lays down short RNA oligonucleotides so

that DNA pol III can bind– RNA is later removed and replaced by DNA

nucleotides (via DNA pol I)

EXTENSION: adding complimentary bases

• DNA Polymerase III is major enzyme• 50 nucleotides/second/replication fork• Each strand acts as a template for making a new

double strand of DNA– Each half (strand) of the original DNA is known as a parent

strand – The new, complementary strand of DNA that matches is

called the daughter strand

DNA Polymerase is 1-directional• LEADING STRAND• Made 5’ 3’

continuously• Follows replisome as

unwound• 1 RNA primer necessary

at start

• LAGGING STRAND• Made 5’ 3’

discontinuously• Short segments =

Okazaki fragments• Goes in opposite

direction to replisome• Uses several RNA

primers• Short segments ligated

afterwards

SEMI-CONSERVATIVE REPLICATION

• ½ old DNA coding for ½ new DNA is known as SEMI-CONSERVATIVE REPLICATION

• During replication: each old double strand will:1. Unzip into 2 single strands, which will:2. Code for a complementary strand (A-T; G-C)3. Which will attach with H+ bonds to form:4. 2 new double strands with:5. 1 new single strand and 1 old single strand each

Semi-Conservative Replication• http://www.dnaftb.org/dnaftb/20/concept/index.html

FYI: Sun directly damages DNA by bonding thymine bases side-by-side. This can create confusion during replication. Polymerase hasdifficulty reading this abnormality and might skip it, match just one A or interpret as a bigpurine.

DNA Repair• DNA pol - error rate of 1 in 10,000 bases

– However proofreading minimizes this to 1 in 10mil

• Mutation any change in DNA– Causes: Genetic error, environmental factors

• Excision Repair – recognize mismatched bp, break sugar-phosphate backbone, remove. DNA pol replaces and ligase connects

Recap…• What enzyme unzips the DNA helix?• What enzyme reads and matches nucleotides

to the parent DNA strand?• When does DNA replication happen?• What is a replisome?• If the parent strand read: ATCGGGTCGT what

would the compliment be?

http://www.bioteach.ubc.ca/TeachingResources/MolecularBiology/DNAReplication.swf

www.johnkyrk.com