DNA Ligase• Energy-dependent joining of the chains

• Activated by NAD+ or ATP hydrolysis NAD NMN+ + AMP

ATP AMP + PPi

• AMP -attaches to lysine group on enzyme

• AMP transferred to 5’ phosphate at ligation site

• 3’ OH at ligation site splits out AMP and joins to 5’ phosphate

Ligase Mechanism

NH2-Lysine-

N

CONH2

O

OH

CH2OO-P-O-P-

OO

OOO CH2

N

N

N

N

NH2

OHHO HO

+

O-P-

O

OO CH2

N

N

N

N

NH2

OHHO

NH2-Lysine-

Activated Phosphorylatingcomplex

NAD+

NMN+

High EnergyNitrogen PhosphateBond

High EnergyNitrogen PhosphateBond

O-P-

O

OO CH2

N

N

N

N

NH2

OHHO

NH2-Lysine-

PO

O

O

OOH

NH2-Lysine-

PO

O

O

OOH

O-P-

O

OOCH2

N

N N

NNH2

OH

HO

OO

OO

P

+ AMP

Eukaryotic Chromosomes have Telomeres

Rule: The lagging strand at the extreme 5’ end of linear chromosomes cannot be accessed by DNA polymerase

Solution: Devise a method to extend the 5’end consistingbasically of non-sensible DNA, to extend the end

TelomeraseAction

Filled in later byDNA polymerase

1. Enzyme binds to TTG

2. Using enzyme’s RNA templateand polymerase, extends3’end of lagging strand

RNA

3. Shifts position to increaselength of lagging strand

Lagging strand

The Legacy of Telomeres

• Gradual loss foretells cell death

• Maintenance signals cell immortality

• Most normal cells have no telomerase

• Cancer cells have telomerase

• Progeria, premature aging, associated with low telomerase activity

• Aging in general may be telomere-related

Reverse Transcriptase

RNA retroviruseseg., HIV, breast cancer

Used to make DNAfrom an RNA template

Major cloning tool

Product is called acDNA (complimentaryDNA)

DNA REPAIRDNA REPAIR

Why the Need for DNA Repair• Chemical modification

• Alterations in the H-bond donor-acceptor pattern

• UV damaged DNA and thymidine dimers

• Methylation and alkylation of DNA

• Point mutationsTransitions and Transversions

• Insertion/deletion mutations

Types of Damage

Cytosine

N

N

NH2

O

Uracil

N

N

O

O

HNH4+

O2 N

N

O

O

HCH3

Thymine

C G U G Corrected by removing U

U A If not corrected

Oxidative

Oxidative deaminations can occur from nitrous acid,derived from sodium nitrite, used as

a food preservative

Oxidative deaminations can occur from nitrous acid,derived from sodium nitrite, used as

a food preservative

UV Radiation

Methylation CH3

CH3

CH3 Only onein vertebratesMethyltransferases

protect bacterial DNA from their restriction nucleases

Methyltransferasesprotect bacterial DNA from their restriction nucleases

Methylation distinguishesparental strandfrom daughterstrand

Methylation distinguishesparental strandfrom daughterstrand

Rule: 80% of human cancers are caused by carcinogensthat damage DNA or interfere with replication or repair

How can one spot a mutagen?

1. Animal StudiesLong and inconclusive

2. Ames TestHistidine deficient Salmonella typhimurium (his-)Add suspected mutagenAdd liver extract Test for spontaneous revertants to (his+)

Two Ways to Maintain a Stable DNA

A replication process of high accuracy

One error for every 108-1010 bases incorporated

Correcting Genetic information when DNA is damaged or modified chemically

Proofreading by 3’- 5’ exonucleases

Chemical modification of nucleotides

Photochemical changes

Correcting Post-Replication Errors in DNAor

DNA RepairDNA Repair

Rule: The repair of DNA is a continuous ongoing event that is linked to a cell surviving free of mutagenicalterations

FACT: An estimated 10,000 bases are set free in DNA every day through breakage of glycosidic bonds

FACT: An estimated 10,000 bases are set free in DNA every day through breakage of glycosidic bonds

Rule: There are many, many ways to repair damage to DNA

Rule: Repair mechanisms tend to overlap and vary in their efficiency and effectiveness of repair

Examples of redundancy: Repair of thymidine dimers

1. Photolysis enzyme: Reduction and bond splitting

2. UvrABC endonuclease: nucleotide excision

Why Overlap?

1. Because photolysis enzyme is designed to spotthe dimer through interaction with active site onthe enzyme.

2. UvrABC is designed to recognize alterations inthe helix structure and thus excises the bases in thearea around any nucleotides that distort the DNA

Types of DNA Repair

1. Direct Repair: Intact Repair (no phosphodiester bonds are broken during the repair)

Examples:

O6-methylguanine-DNA methyltransferase

O

N

NNH2 N

NH

CH3 O

N

NNH2 N

NH

Enz-SH Enz-S-CH3

inactiveactive

Excision Repair Enzymes

• UvrABC endonuclease (helix distortions)

• DNA glycosylase (damaged base)

• AP endonuclease (missing base)

• Uracil N-glycosylase (uracil in DNA)

Excision RepairUvrABCendonuclease DNA glycosylase

Pol I

Double Strand Breaks - Recombination - Error Prone

Rule: When both strands of DNA are damaged, excision repair has no means to gauge a repair

Rule: Interruptions in the movement of the replicatingfork elicit a higher order repair system called SOS

Rule: Recombinatorial DNA repair or error-pronerepair is activated whenever DNA damage occurs ata high level….this is the SOS response

SOS and Recombination Repair

• Occurs when damaged DNA is being replicated

• Controlled by RecA and LexA

• Error prone

• No template to guide

• Operates by genetic recombination via RecA

SSDNA-RecA

Hydrolyzed LexA

mRNA for SOS repair enzymes + mRNA for LexA and RecA

Turned on

LexA repressor SOS genes

uvrA,B,ClexA recA

RecombinatorialRepair

Error-proneRepair

SOS

Mechanism of RecA in Recombination Repair

1. Formation of RecA filaments

2. Alignment with homologous dsDNA

3. Unraveling and binding the replacing strand

4. ATP-dependent repair

5. Displaced intact ssDNA template