Chapter 11 - DNA replication and recombination

DNA replication underlies the process of

inheritance at all levels

many cells do not undergo

replication

DNA replication occurs as a prelude to

cell division

_ of DNA strands allows each strand to serve as a template for synthesis of the other

complementarity

DNA replication is also based on the ability of proteins to form _ with specific sequences of DNA

specific interactions

there are three modes of DNA replication

semiconservative, conservative, dispersive

semiconservative is when each replicated DNA molecule consists of

oneone old and one new strand

conservative - come together and the original is -

two newly synthesized strands, conserved

dispersive parental strands are dispersed into

two new double helices

Meselson-Stahl experiment

used radioactive 15N nitrogen and 14N nitrogen to differentiate old and new DNA

Meselson-Stahl used equilibrium density gradient centrifugation to separate DNA containing , DNA containing _*,* and DNA containing _

all 15N, half 14 and 15N, all 14N

the old DNA would only contain

15 N

the group of DNA that replicated once would contain

half 14 N and half 15N

the group of DNA that have replicated twice would contain some

DNA with only 14N and some DNA with both 14N and 15N

the thickness of each band denotes the

amount of DNA that has each isotope

the result of Meselson-Stahls experiment ruled out conservative model because

it predicted that there should be two bands

to distinguish between the semiconservative model and the dispersive model,

E. coli were grown for another generation

dispersive model predicted that there should only be _, therefore it was ruled out

one band

Autoradiography pinpoints location of

radioisotope in cell

from autoradiography, we can identify the location of

newly synthesized DNA

Vicia faba was used to demonstrate DNA replication is

semiconservative in eukaryotes

DNA synthesis in bacteria involves five polymerases

DNA pol I, II, III, IV, V

DNA polymerases catalyze the formation of a

phosphodiester bond between the 3’ OH and 5’ phosphate

the energy for the formation of a phosphodiester bond is supplied by the

hydrolysis of the two phosphates from the dNTP

all DNA polymerases synthesize DNA in the

5’ to 3’ direction

all DNA polymerases require a

template

DNA polymerases are

fast

chain elongation is catalyzed by DNA polymerase _

III

DNA polymerase I is isolated from

E. coli

DNA polymerase I repairs DNA and participates in synthesis of the

lagging strand during replication

DNA polymerase I requires

DNA template, all four deoxyribonucleoside triphosphates, and primer

DNA polymerase I, II, III can elongate existing _ but cannot initiate _

DNA strand (primer), DNA synthesis on its own

DNA polymerase I, II, III have

exonuclease activity 3’ - 5’

exonuclease activity 3’ - 5’ is the of newly synthesized DNA, _

proofreading, removing and replacing incorrect nucleotides

only DNA polymerase I has

exonuclease activity 5’ - 3’

exonuclease activity 5’ - 3’ allows DNA polymerase to remove DNA or RNA from and excises _, filling in the gaps left behind

5’ end of a molecule, primers

DNA polymerases I, II, IV, V are involved in various aspects of

DNA repair

holoenzyme : active form of

DNA pol III

holoenzyme is an asymmetric dimer made up of

core enzyme complexes, sliding clamps and a single y complex

in holoenzymes, core enzyme subunits each have separate functions

alpha, epsilon, and theta

alpha is involved in

5’ - 3’ polymerization

epsilon is involved in

3’ - 5’ exonuclease activity (proofreading)

theta is involved in

core assembly

DNA polymerases uses sliding ring clamps to

increase processivity

processivity is an enzyme’s ability to catalyze

consecutive reactions without releasing and rebinding its substrate

processivity due in part to dimer of beta subunits, which form a sliding clamp, locking the active site of

DNA pol III into the 3’ end of the growing chain

DNA replication begin at the ORI, an , where the helix is _ to form a

AT rich region, replication bubble

there are two replication forks at either end of a bubble, where occurs, using each separated strand as a _

DNA synthesis, template

replication is bidirectional, replication bubble grows as the two replication forks move in

opposite directions

Dna A unwinds the

helix

Dna A binds to ORI causing , causing the helix to _

conformation change, destabilize and open up

Dna A exposes

ssDNA

DNA helicase is made of

DnaB polypeptides

DNA helicase is a _ of subunits, assemble around _

hexamer, exposed ssDNA

helicases require energy supplied by hydrolysis of ATP to untwist the DNA ahead of *_* and denatures hydrogen bonds and destabilizes _

polymerase III, double helix

ssBPs =

single stranded binding proteins

ssBPs _ the open conformation of helix and bind specifically to _

stabilize, single strands of DNA

ssBPs prevents DNA from reforming _ (reannealing) and protects ssDNA from

double stranded regions, hydrolysis by nucleases

ssBPs also make DNA inflexible and better substrate for

base pairing

ssBPS are displaced by

DNA pol III

_ relieves coiled tension from unwinding of helix

DNA gyrase

DNA gyrase is a member of larger enzyme group:

DNA topoisomerase

DNA gyrase makes _ and is driven by energy released during _

single or double stranded cuts

primase is

RNA polymerase

primase is _ to replication fork by helicase and synthesizes _

recruited, RNA primer

primase provides 3’ OH required by _ for elongation

DNA polymerase III

RNA priming is a universal phenomenon and is found in

bacteria, viruses, and several eukaryotic organisms

proofreading and error correction is an integral part of

DNA replication

DNA polymerase exonuclease activity of 3’ - 5’ allows for

excise of nucleotides

there are three distinct stages of DNA replication

initiation, elongation, termination

in bacterial replication, their chromosomes only have

one ORI

bacteria DNA synthesis originates at Ori C and proceeds

bidirectionally

replicon is the length of

DNA replicated

initiation of replication starts with the binding of an _ which denatures the oriC and then recruits a - which - the DNA in both direction

initiator protein DnaA, DNA helicase, untwist

next, each helicase recruits a DNA primase to form a

primosome

DNA primase makes the necessary RNA primers needed by

DNA polymerase III

after RNA primers are made, the _ that are involved in DNA replication are assembled

proteins

replisome : protein machine that carries out -, all proteins at work the -

polymerization reaction, replication fork

two replisomes per -, one per -

genome, replication fork

replisome is made out of the - and -

DNA pol III holoenzyme, primosome

enzymes and proteins are essential to DNA synthesis

DNA polymerase III core enzymes, ssBPs, DNA gyrase, DNA helicase, RNA primer

two strands of double helix are -: 5’ - 3’ and 3’ - 5’ but DNA pol III only synthesizes -

antiparallel, 5’ - 3’

DNA pol III synthesizes two strands simultaneously but in the

opposite direction

continuous DNA synthesis: leading strand = made in the - as fork movement; only

same direction, one primer required

discontinuous DNA synthesis: lagging strand = new strand formed by polymerization - from replication fork; -required

away, many primers

DNA strands at a single replication fork are synthesized

concurrently

the lagging strand is looped, inverting the

physical but not biochemical direction

DNA polymerase III has two core complexes

one core complex synthesizes the leading strand, the other synthesizes the lagging strand

DNA pol III dimer initiates polymerization by - to the - on each not the strands

adding dNTPs, RNA primer

DNA clamp prevents core

enzyme dissociation from template

lagging strand synthesized as -, each with RNA primer

Okazaki fragments

DNA polymerase I removes - on lagging strand using - activity and fills in gaps

primers, 5’ to 3’ exonuclease

DNA polymerase I is a single polypeptide with

three separate active sites

DNA polymerase III ends polymerization when it encounters

double stranded DNA ahead

when DNA polymerase III dissociates from the DNA, it leaves a gap in one strand, this gap is recognized as - and is repaired by -

damaged DNA, DNA polymerase I

DNA ligase seals nicks and -, catalyzing formation of -

joins fragments, phosphodiester bonds

termination of replication occurs at - (ter), which has binding sites for a protein known as - (tus)

termination site, terminator utilization substance

tus prevents replication forks from passing through region by inhibiting the

helicase of the replisome

eukaryotic and bacterial DNA replication

shares many features

eukaryotic DNA replication is more complex because there is - than prokaryotic cells, - chromosomes, DNA complexed with -

more DNA, linear, nucleosomes