Biochem Chapt 25: Dna Metabolism

Front 1

What is DNA metabolism?

Back 1

Consist of tightly regulated processes that achieve:

A new copy of dna to fidelity before each cell division

Repairs made to errors in dna synthesis

Novel dna created through Recombination (segments of 2 dna molecules rearranged) or rearrangement within chromosome

Front 2

3 rules of dna replication

Back 2

1. Replication is semiconservative

2. Begins at an origin and proceeds bidirectionally (usually)

3. Synthesis of new dna occurs in 5-3’ direction and is semi-discontinuous

Front 3

Semi-conservative replication

Back 3

Meselson-stahl experiment: N is divided equally between 2 daughter genomes

So each new strand has one parent and one new daughter strand

Front 4

Meselson-stohl experiment

Back 4

Used Heavy N (15) for one strand and light N (14) for another and allowed cells to divide

Found one heavy and one light (parent and daughter) strand

Allowed cells to divide again and had 2 of the hybrid plus 2 that were all light

Front 5

DNA is bidirectional (cairns experiment)

Back 5

Showed in bacterial dna that both strands replicated simultaneously

2 replication forks suggested bidirectional replication

Front 6

Can replication begin anywhere? (Inmans experiment)

Back 6

Found dna always started at A-T rich regions forming bubbles.

These bubbles were mapped (denaturation mapping) and found loops initiated at unique points and called them origins

Front 7

Leading vs lagging strand

Back 7

Synthesize dna in 5->3’ direction

Because one parent strand is in 3->5’ direction, it is easier to replicate and is called the leading strand (does it faster so leads) and is continuous

The other parent strand is in 5->3’ so the daughter strand is made in small segments to overcome which takes time (lagging “behind” strand) and is discontinuous due to Okazaki segments

Front 8

Enzymes that degrade dna segments

Back 8

Nucleases degrade nucleic acids (DNases degrade dna and RNases degrade rna)

Exonucleases cleave bonds that remove nucleotides from ends of dna

Endonucleases cleave bonds within DNA sequence

Front 9

Enzymes that create dna segments

Back 9

Polymerases

DNA polymerase I was discovered by kornberg

Front 10

Chemistry of dna elongation

Back 10

Nucleoside triphosphates serve as substrates in strand

OH acts as a nuclephile

It is activated via Mg by deprotonating the OH group making it a better nucelophile

The OH attacks the alpha phosphate at the 3’ end of the growing chain

Pyrophosphate is a good leaving group

Front 11

DNA polymerase requires a primer

Back 11

A short strand complementary to the template

Contains a 3’ OH to begin the first DNA polymerase-catalyze rxn

Can be made of dna or rna (more common)

Front 12

DNA polymerase

Back 12

Enzyme that has a pocket with 2 regions:

Insertion site- where incoming nucleotide binds

Postinsertion site- where the newly made base pair resides when the polymerase moves forward

Front 13

Processivity

Back 13

The number of nucleotides added by DNA polymerase before it dissociates

Processivity varies among DNA polymerases (each has its own and each also has its own polymerization rate)

Can vary from a few to thousands

Front 14

Geometry of base pairing accounts for high fidelity

Back 14

DNA polymerase active site excludes pairs with incorrect geometry

Sometime will insert wrong one but has ways to fix these errors

Front 15

Errors during synthesis are corrected by 3->5’ exonuclease activity

Back 15

All DNA polymerase have an additional activity

Exonuclease “proofreads” synthesis for mismatch base pair

Translocation of enzyme to next position is inhibited until enzyme removes incorrect nucleotide

Front 16

In e. Coli there are at least 5 dna polymerases

Back 16

DNA polymerase I is abundant but not ideal for replication bc it has low processivity

It’s primary function is clean up

DNA polymerase III is the replicator

II, IV, and V are involved in dna repair

Front 17

Klenow fragment

Back 17

A distinct domain that can be separated by protease tx

DNA polymerase I has additional 5->3’ exonuclease activity that moves ahead of the enzyme and hydrolyze a things in its path

Nick translation-a strand break moves along with enzyme

Front 18

Nick translation

Back 18

A break in strand

Replace a segment

Front 19

DNA polymerase III complex

Back 19

Has:

3 cores with beta subunits-beta subunits increase processivity of the complex

Clamp loader- clamps on dna and then feeds through cores

Front 20

Replisome

Back 20

A set of 20 enzymes and proteins that are needed for dna replication in e.coli

Includes:

Helicases-use atp to unwind dna strands

Topoisomerases- relieve stress caused by unwinding

DNA-binding proteins- stabilize strands

Primase- make rna primers

Dna ligases- seal nicks btw Okazaki fragments

Front 21

Initiation of replication in e.coli

Back 21

Begins at oriC site

A-T rich region

Front 22

Proteins and steps in initiation of replication in e. Coli

Back 22

DNA Wraps around dnaA protein complex forming a supercool which leads to denaturation of DUE (dna unwinding element) sites

DnaB migrates along 5-3’ and unwinds helix (helicase)

Other proteins link to dnaB

Single-stranded dna binding protein (SSB) stabilizes strands

DNA gyrase relieves tension ahead of forks

Front 23

Regulation of initiation via methylation

Back 23

Dam (dna adenine methylase) methylase hemimethylates oriC so it can interact with plasma membrane

Allows new dna to bind

Front 24

Elongation of leading strand

Back 24

Straightforward approach

Primase (dnaG) makes rna primer (interacts with helicase but moves in opposite direction)

DNA polymerase III adds nucleotides to the 3’ end of the strand

Front 25

Elongation of the lagging strand

Back 25

RNA primer and DNA polymerase III are the same

Synthesis proceeds with nucleotides adding 3’ end

Elongated away from fork

One DNA polymerase III dimer complex synthesizes both strands

Front 26

Leading and lagging synthesis

Back 26

Core subunits of DNA polymerase III dissociate from on beta clamp and bind a new one

RNA primer is removed by DNA polymerase I or rnase H1

DNA polymerase I fills the gap

DNA ligand seals backbone

Front 27

3 rd stage of replication is termination

Back 27

Replication forks meet at Ter sites found near each other in opposite directions

Ter is binding site for Tus (terminus utilization sequence) that cause a replication fork to stop

Front 28

Replication in eukaryotes

Back 28

More complex than bacteria

Yeast have 400 origins so called autonomously replicating sequences (ARS)

Entire genome replicated 1x/cell cycle

Regulation due to cyclin dependent kinases (CDK) which are ubiquinated for destruction at end of mitosis phase

Front 29

Initiation of replication in eukaryotes

Back 29

Require a pre-replication complex (pre RCs)

Origin recognition complex (ORC) loads a helicase onto DNA (like bacterial dnaA)

Helicase is a hexamer of mini chromosome maintenance protein (like dnaB)

Occurs more slowly than bacteria (50 nucleotides/sec)

Front 30

Multiple DNA polymerases in eukaryotic replication (3 primary mulitsubunit)

Back 30

1. DNA polymerase alpha:

has primase activity in one subunit and polymerization in another

Does not have 3-5 proofreading

2. DNA polymerase E:

synthesizes leading strand

Highly processive and has proofreading

3. DNA polymerase &:

Synthesis for lagging strand and has proofreading

Front 31

Termination of replication in eukaryotes

Back 31

Synthesis of telomeres (found at ends of chromosomes)

Enzyme telomerase uses rna

Front 32

DNA repair and mutations

Back 32

Damage to dna genome -majority are fixed but some escape repair changing daughter dna

Accumulations of mutations is correlated to cancer

There are 1,000 of lesions daily (Unrepaired dna) but only 1/1000 lead to mutations

Human genome contains genes for over 130 repair proteins

Front 33

DNA lesions and mutations

Back 33

A lesion is dna damage

If unrepaired, leads to mutation

Mutations can be substitutions (point mutations), deletions, additions, and silent mutations (have no effect on gene functioning)

Front 34

Type is dna damage

Back 34

Mismatch (from incorrect nucleotides)

Abnormal bases from deamination, alkylation, free radicals

Formation of pyrimidines due to UV light exposure

Backbone lesions from radiation and free radicals

Front 35

Mismatch repair in e. Coli

Back 35

One parent strand is methylated ( DAMmethylase) insert CH3

The new daughter strand is unmethylated for a short period of time after synthesis (any errors will reside here)

The methyl -directed mismatch repair system will cleave the unmethylated strand in the initial part of the repair process

Front 36

Methylation-directed repair in e.coli

Back 36

See pic

Front 37

Base excision repair

Back 37

Uses dna glycosylases (recognize specific lesions and cleave between sugar and base)

Uracil glycosylase removes uracil (C deaminates to U and U is in RNA)

Front 38

Base excision repair in depth

Back 38

Nucleotide or region is removed by AP endonucleases

Then DNA polymerase I adds new dna

DNA log see seals Nick

Front 39

Nucleotide excision repair

Back 39

Lesions include: pyrimidine diners, photoproducts (UV light damage for both of those), benzopyrenguanine (from cigarette smoke)

Removal of dna by excinucleases

Front 40

Direct repair includes:

Back 40

Photolyases (but don’t have this)

O6-methylgaunine-dna methyltransferase (repairs methylated gaunine)

AlkB demethylates 1-methylase nine and 3- methylcytosine

Front 41

If there is no undamaged dna to use as a template, we use

Back 41

Another chromosome (recombination)

Or

Error-prone translesion synthesis (TLS)

Front 42

Error prone tranlesion

Back 42

In bacteria: sos response when dna damage is extensive (involved DNA polymerase V)

In mammals: TLS polymerases that recignize specific types of damage and respond (dna n when t-t dimer halts fork)

Unmutable

Front 43

DNA recombination

Back 43

Segments of dna can rearrange within chromosomes from one to the other

Involved in repair of dna, segregation of chromosomes during meiosis, and increase in genetic diversity

Driving force of evolution along with mutations

Helps virus and bacteria develop resistance

Front 44

3 classes of recombination

Back 44

Homologous: exchange btw dna that share a region of similar sequence

Site-specific: exchange only at a particular sequence

DNA transposition: jumping genes

Front 45

Homologous replication in eukaryotes

Back 45

Occurs frequently during meiosis

Crossing over in first meiotic division at chiasmatas where dna breaks and rejoins (called hot spots also)

At the end of meiosis II: sister chromatids are separated with different genetic material inceasing genetic diversity

Front 46

Crossing over/ recombination

Back 46

See pic

Front 47

Nonhomologous end joining

Back 47

Another way to repair

Not ideal

Broken chromosomes ends are lighted back together

No conservation of DNA sequence

Front 48

Site-specific recombination

Back 48

Limited to specific sequences with either Ter or Ser

Recombinase cleaves strand and joins with new partners

Front 49

Transposable genetic elements (transposons)

Back 49

DNA that carries genes for transposases can move to a nee region or chromosome

Cut themselves out and insert elsewhere and then these sites can be duplicated

Ie: develop antibiotic resistance