L14 DNA Replication, Repair, and Recombination

DNA-Protein Interactions

-Many proteins bind DNA nonspecifically

-Generally do so weakly

-Many proteins bind DNA with high sequence specificity despite the sequence independent structure of B-DNA

-Typically do so without distorting DNA base pairing

-Proteins bind to the base specific functional groups that are exposed in the major and minor grooves

-Typical Kd's of 10^-9 to 10^-12

*idk wtf any of this means

Zinc finger

-~30 residue modules

-Typically present in tandem

-Contain Cys2-His2Zn2+ binding site

=Binds 3-bp segments in the major groove

Leucine Zipper Proteins

-Leu zipper motifs mediate dimerization of DNA-binding motifs

-Heptad repeats that form coiled coils

-Leu every 7th position

Prokaryotic chromosomes

-Circular DNA molecule that is extensively looped and coiled

-Supercoiled DNA complex with a protein core core in structure called the nucleoid

-Proteins in center hold superhelical loops

-Bacteria typically have singular circular chromosome

Eukaryotic chromosomes

-Chromosome number and length can vary by species

-Each eukaryotic chromosome consists of a singular, linear DNA molecule complexed with histone proteins to form necleosomes

-chromatic is the term used to describe this complex

What are the five major classes of histones?

H1, H2A, H2B, H3, and H4

What is a nucleosome?

A negatively supercoiled DNA wrapped around a histone core (two copies of each of H2A, H2B, H3, and H4)

What part of the histones protrude from the nucleosome?

The N-terminal tails of the histones protrude from the nucleosomes and cam be covalently modified (methylation and acetylation)

*a type of epigenetic modification

Octamer

8 total peptides within the (two copies of each of H2A, H2B, H3, and H4)

What binds to the outside to stabilize?

H1

Are histones positively or negatively charged

positively

How many base pairs and how many turns?

~166 base pairs and 2 turns

For cell division chromatid must be what?

compacted into chromosomes

-nucleosomes coiled into 30nm fiber, then further coiled into 200nm filaments

During interphase, chromatin is in one of two forms...

-heterochromatin (highly condensed)

-euchromatin (less condensed)

DNA Replication is always_____. And never_____ or _____.

DNA replication is always semiconservative (simultaneously make copies of both original strands) and never conservative (copy on strand. Use first strand copy to make other new strand) or dispersive (copied DNA is interspersed with both original strands.

Describe the Meselson-Stahl Experiment.

-Growing bacteria on 15N medium adds about 1% to the mass of each base-pair

-Can be distinguished using density gradient centrifugation of the DNA

-Results were consistent with expectations for semi-conservative DNA replication and conclusively disproved conservative and dispersive DNA replication

-Semi-conservative replication must appear if the two parental strands (top) are separated . This produces the replication fork

*essentially proved the DNA was separated before replication. which supported semi-conservative replication

The DNA Polymerase Reaction

-Extension is from 5' to 3'

-A primer is required

-Pyrophosphate is produced

-Added nucleotide contributes the PO4

-The reaction requires a free 3' hydroxyl

-Of incoming nt is not perfectly base-paired, the extension will not occur

-If template end is not base-paired it is hydrolyzed off (proofreading activity.)

Leading strand

synthesis follows opening of the fork

Lagging strand

synthesis cannot start until the fork has moved some distance, and so is made in short segments called Okazaki fragments

-1000-2000 nucleotides in prokaryotes

-100-200 nucleotides in eukaryotes

The DNA Polymerase Reaction Mechanism

-All DNA (and RNA) polymerases use the same mechanism

-In-line necleotidyl transfer

-3' hydroxyl nucleophile on alpha-phosphate group of incoming dNTP

-Two Mg2+ ions (A and B)

-activate the nucleophil

-neutralize the negative charge

-stabilize phosphate leaving group

*idk wtf any of this means

In what direction does Pol 1 have exonuclease activity

-in both 3'-->5' and 5'-->3' directions

What occurs when an incorrect base is added?

-polymerase activity stalls

-3'-->5' exonuclease activity hydrolyses last added mono nucleotide

What happens when Pol 1 binds to duplex DNA at single strand nicks?

-5'-->3' exo activity hydrolyses up to 10 nt

-Pol 1 polymerase activity fills in

-nick translation

-nick is sealed by DNA ligase

DNA Replication in Prokaryotes

-DNA unwinding

-Primer synthesis

-DNA synthesis

-Joining DNA fragments

DNA unwinding

-requires helicases, which are ATP-dependent enzymes that catalyze the unwinding of duplex DNA (e.g., DnaB in E. coli)

Primer synthesis

-is the formation of short RNA segments (primers) required for the initiation of DNA replication by primase (e.g., dnaG)

DNA synthesis

-of complementary DNA is performed in a 5'-->3' direction and catalyzed by a large multi-enzyme complex referred to as DNA polymerase III holoenzyme

Joining DNA fragments

-frequently during DNA synthesis, DNA segments must be joined together

-DNA polymerase I: removes RNA primers and replaces with RNA

-DNA ligase: catalyzes the formation of the phosphodiester bond between adjoining nucleotides

DNA Polymerase Senses Proper Base Pairing

-Only conformation samples available dNTPs

-Only when complementary dNTP binds does the catalytically active close form occur

-Distortions with mismatched base would make the closed form catalytically inactive

(induced fit)

What does helicase do?

-Unwind the DNA

-Driven by NT hydrolysis

What is the replisome?

-The DNA replicating machine

-consists of 2 pol. III holoenzymes, the primosome, and DNA unwinding proteins.

-The DNA polymerases (with sliding clamps) are associated as a dimer and are oriented in the same direction

-the lagging strand must loop around to make this posssible

-The loop grows and shriks with generation of each fragment (resembles a trombone slide?)

What controls supercoiling

-DNA topoisomerases

-Relieve torque in DNA

-DNA gyrase in E.coli

Type II topoisomerase produces transient double-strand breaks

ssDNA

-unwindind generates ssDNA

-not stable

-prefers to reanneal to complement

-To maintain ssDNA, specific proteins (singl-stranded DNA-binding SSB proteins) bind

DNA Polymerase III

-major replicative polymerase in prokaryotes

-composed of at least 10 subunits

-The core polymerase is formed of three subunits (alpha, epsilon, and theta)

-The B-protein (sliding clamp) is two subunits and forms a donut-shaped ring around the template DNA

-B2-Clamp promotes processivity (prevents dissociation of polymerase from the DNA template)

>5000 residues vs 12 residues

-The gamma complex acts as the clamp-loader, loading B2-clamp dimer

DNA polymerase I

-involved in RNA primer removal and replacement with DNA

-Contains 3 enzymatic activities

-5' to 3' exonuclease activity (removes RNA primers

-3' to 5' exonuclease activity (proofreading)

-5' to 3' templated DNA polymerase activity (filling in)

DNA polymerase II, IV and V

-involved in DNA repair translesion repair enzymes

-SOS response that prevent cell death dut to high levels of DNA damage

How often is their an error in DNA replication in E. coli

-DNA replication in E. coli is amazingly accurate

-one error per 10^9 or 10^10 base pairs

-Copying mechanism, proofreading, post replication repair

Origin of Replication In Bacteria

-Single initiation site (oriC)

DnaA proteins

-bind to five to eight 9-bp sites withn the oriC

-Results in nucleosome-like structure requiring ATP

-Causes opening of DNA

DnaB

-complex with DnaC centers that open oriC region

-Once DnaB is loaded, DnaC is released

-The replication fork moves forward as DnaB unwinds the helix

-Allows replisome to form and begin replication fork

iClicker Question:

The primers used during cellular DNA replication

are made by an enyzyme that catalyzes a polymerase reaction that does not depend on extending a pre-existing DNA

Timing of replication in Eukaryotes

Eukaryotic replication is limited to a very specific phase of the cell cycle (S phase)

Replication rate in Eukaryotes

~10x slower in eukaryotes at each replication fork due to complex chromatin structure

How many distinct DNA polymerases are there?

at least 13

Which DNA polymerases are involved in nuclear replication?

alpha, delta, and epsilon

Which DNA polymerase replicated and repairs mitochondrial DNA

Polymerase gamma

Which DNA polymerases function in nuclear DNA repair?

beta, zeta and nu

DNA polymerase alpha

-initiating polymerase

-associated tightly with a primase

-complex makes 7-10 per RNA primer and them ~15 DNA bases

DNA polymerase delta

-Lagging strand DNA polymerase

-3'-->5' exonuclease activity

-processivity nearly unlimited with bound to sliding-clamp protein

-proliferating cell nuclear antigen (PCNA)

DNA polymerase epsilon

-likely leading strand polymerase

-3'-->5' nuclease activity

-highly processive without PCNA

PCNA

sliding clamp

MCM

-helicase

-tracks along the leading strand template at the replication fork

-heterohexameric complex

Replication Protein A (RPA)

trimeric protein that binds sing stranded DNA

Primer removal

-No nick-translating polymerasee like pol I

Rnase HI

-cleaves RNA

Flap endonuclease I- (FENI)

-Recruited by PCNA

Gaps filled by DNA polymerase delta

Nucleosomes

-Disassemble just ahead of the replication fork

-Reassemble in daughter duplexes

Initiation of Replication in Eukaryotes

Eukaryotes have multiple replicons

-One every 3-300kb

-Humans have ~30,000 origins of replication

Replication initiates at autonomously replication sequences (ARS)

-conserved 11-bp sequences

-adjacent to easily unwound DNA

Replication begins with the assembly of the pre-initiation replication complex (preRC)

-Begins assembly of the origin recognition complex (ORC)

-Includes the MCM helicase

-Cell cycle kinases phosphorylate and activate preRC components to initiate replication