DNA replication and Repair

DNA and RNADr.S.Sethupathy,M.D.,Ph.D,Professor & Head of BiochemistryRMMCAU

Deoxyribo Nucleic Acid (DNA)Deoxyribose sugarNitrogenous bases Purine bases- Adenine and GuaninePyrimidine bases- Thymine and cytosinePhosphoric acidBase + sugar = nucleosideBase + sugar + phosphoric acid = Nucleotide

DNA

DNA structure

Deoxy nucleosides and nucleotidesDeoxy adenosine, deoxy guanosine, dexoy cytidine, deoxy thymidine

Deoxy adenylate, deoxy guanylate, dexoy cytidylate, thymidylate (dAMP, dGMP, dCMP, TMP)

Double strandedPolarity AntiparallelEqual number of adenine and thymine basesEqual number of guanie and cytosine basesChargaffs base pairing rule A .. T and G C

Sugars

RNA vs DNA

Difference between DNA & RNA

RNA

Transfer RNA

messengerRNA

DNA replication and RepairDr.S.Sethupathy, M.D.,Ph.D.,Professor & HOD of Biochemistry,Rajah Muthiah Medical College,Annamalai University.

DNA replicationCopying of DNA DNA from DNAOnly onceSemi conservativeEach strand acts as templatePolarity is maintained.Reading 3 to 5 Synthesis 5 to 3

Steps of replicationIdentification of OriginUnwinding of DNAReplication fork and RNA primer synthesisSynthesis and elongation of DNA - PolymerizationReplication bubbles and ligation of gapsChromatin assembly formation

Origin of replicationE.coli Dna A protein finds Ori CYeast- Origin Recognition complex (ORC)Eukaryotes - DNA unwinding element (DUE)

Replication forkAs DNA synthesis continues, there is a replication fork with two prongs. Eukaryotes have replication at multiple origins DNA helicase unwinds DNAHelicases - separate strands of a DNA double helix Also remove nucleic acid-associated proteins.

Single-strand binding proteins bind to the DNA (SSBPs) (Gyrase)Topisomerases I and II removes supercoils by nicking and sealing.RNA primer11- 60 nts lengthDna G synthesize RNA polymerase activityLeading strand oneLagging strand many

DNA polymeraseReading 3 to 5Synthesis 5 to 3Leading strand (forward) continuousThe lagging strand is synthesized in short segments as Okazaki fragments.

DNA base pairing

TerminationIn Eukaryotes DNA replication is unable to reach the very end of the chromosomes.The telomere of the daughter DNA strand shortens. So somatic cells can only divide a certain number of times. In germ cell line, telomerase enzyme extends the repetitive sequences of the telomere region to prevent degradation. Telomerase if active in somatic cells, leads to cancer.

DNA ligaseDNA ligases close nicks in the phosphodiester backbone of DNA. DNA ligases are essential for joining Okazaki fragments .Involved in DNA repair process.

Reverse transcriptaseIn retroviruses, the RNA is the template. Reverse transcriptase DNA from RNADNA-RNA hybrid is synthesized RNAase H degrades the RNA Then dsDNA is synthesized from DNA template.Retroviruses (e.g., HIV), and RT inhibitors are widely used as antiretroviral drugs.

27

Cell cycle

Clinical applicationsA disregulation of the cell cycle lead to tumor formation.Cell cycle inhibitors, RB, p53 etc. on mutation, form a tumor.G1 is the most variable phase of the cell cycle.

DNA repair DNA damage needs repair. Ineffective repair lead to three possible states:1. an irreversible state of dormancy, known as senescence2. cell suicide, also known as apoptosis or programmed cell death3. unregulated cell division, which can lead to the formation of a tumor that is cancerous

Types of DNA damage

1. Endogenous damage - attack by reactive oxygen species produced from metabolism.2. Exogenous damage by external agents ultraviolet , X-rays and gamma raysThermal disruption, toxins, mutagenic chemicals, aromatic compounds that intercalate DNA viruses.

1.Single base alteration 2.Two base alterationa.UV light induced thymine- thymine dimerb.Alkylating agent cross linkage3. Chain breaksIt is due to ionizing radiation, radioactivity, oxidative free radicals.4. Cross linkage

The mechanisms of DNA repair

1. Nucleotide excision repair (NER)2. Mismatch repair (MMR)3. Base excision repair (BER)4. Homologous recombination repair (HRR)5. Non homologous end joining (NHEJ)

Double strand breaks

Transcription

Synthesis of RNA from DNAReading of DNA from 3 to 5Synthesis of RNA from 5 to 3RNA polymerasePromoter region regulates transcriptionSteps- Initiation , elongation, termination

Transcription

Transcription RNA from DNA

Translation RNA to proteinsSynthesis of proteinsmRNA contains the message in the form of codonstRNA carries amino acids and recognize codons with its anticodonrRNA is involved in translation