DNA Ligase Energy-dependent joining of the chains Activated by NAD + or ATP hydrolysis NAD  NMN + + AMP ATP  AMP + PP i 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 NH 2 -Lysine- + O-P- O O O CH 2 N N N N NH 2 OHHO Activated Phosphorylating complex NAD + NMN + High Energy Nitrogen Phosphate Bond High Energy Nitrogen Phosphate Bond

O-P- O O O CH 2 N N N N NH 2 OHHO NH 2 -Lysine- P O O O O OH

NH 2 -Lysine- P O O O O OH O - P - O O O C H 2 N N N N N H 2 O H H O O O O O 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 consisting basically of non-sensible DNA, to extend the end

Telomerase Action Filled in later by DNA polymerase 1. Enzyme binds to TTG 2. Using enzyme’s RNA template and polymerase, extends 3’end of lagging strand RNA 3. Shifts position to increase length 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 retroviruses eg., HIV, breast cancer Used to make DNA from an RNA template Major cloning tool Product is called a cDNA (complimentary DNA)

DNA 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 mutations Transitions and Transversions Insertion/deletion mutations

Types of Damage Cytosine N N NH 2 O Uracil N N O O H NH 4 + O2O2 N N O O H CH 3 Thymine CGCGUGUGCorrected by removing U UAUAIf 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 CH 3 Only one in vertebrates Methyltransferases protect bacterial DNA from their restriction nucleases Methyltransferases protect bacterial DNA from their restriction nucleases Methylation distinguishes parental strand from daughter strand Methylation distinguishes parental strand from daughter strand

Rule: 80% of human cancers are caused by carcinogens that damage DNA or interfere with replication or repair How can one spot a mutagen? 1. Animal Studies Long and inconclusive 2. Ames Test Histidine deficient Salmonella typhimurium (his - ) Add suspected mutagen Add 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 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 DNA or DNA Repair Rule: The repair of DNA is a continuous ongoing event that is linked to a cell surviving free of mutagenic alterations 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 spot the dimer through interaction with active site on the enzyme. 2. UvrABC is designed to recognize alterations in the helix structure and thus excises the bases in the area 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: O 6 -methylguanine-DNA methyltransferase Enz-SH Enz-S-CH 3 inactiveactive

Excision Repair Enzymes UvrABC endonuclease (helix distortions) DNA glycosylase (damaged base) AP endonuclease (missing base) Uracil N-glycosylase (uracil in DNA)

Excision Repair UvrABC endonuclease 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 replicating fork elicit a higher order repair system called SOS Rule: Recombinatorial DNA repair or error-prone repair is activated whenever DNA damage occurs at a 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

SS DNA-RecA Hydrolyzed LexA mRNA for SOS repair enzymes + mRNA for LexA and RecA Turned on LexA repressor SOS genes uvrA,B,ClexArecA

Recombinatorial Repair Error-prone Repair 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