Topic 6 DNA Replication and Repair

DNA replication

genome accurately copied before cell division

Template

strand of DNA, used to synthesis complmentary strand, relies in complementary base pairing

Replication machine

proteins that carries out protein replication

semiconservative model

daughter DNA contain one parental strand and one newly synthesized strand

Replication origin

location where replication begins, ~100 nt long, bacteria-one replcation origin (1 circular chromosome), Human genome - 10000origins (220 per chromosomes)

Initiator proteins

bind DNA at replication origin, pry DNA strand apart (break H bond), attracts proteins of replication machine

Replication fork

where dsDNA unwound into ssDNA, 2 forks form (move opposite directions), bidirectional DNA replication

Replication machine

unzip DNA at fork, use DNA as template to make new daughter strand, bacteria - 1000nt per sec, Humans - 100nt per sec (slower bc more complex)

DNA polymerase 3

add nt to 3’ end, catalyze phosphodiester bonds(when conformational change happens), one error every 10^7 nt, has proofreading domain that corrects mistakes

Proofreading of DNA pol 3

correct error, double check work before adding next, different domain than polymerization

Deoxyribonucleoside triphosphate (dNTP)

how nucleotide enter DNA machine, provides own energy for it’s addition, hydrolysis release pyrophosphate

Leading strand

synthesized continuously, move toward replication fork

lagging strand

synthesized discontinuously, move away from replication fork, form okazaki fragments, slight delay compared to leading

RNA primer

~10 nt long, provides 3’ group for DNA pol to add to, leading strand need 1, lagging strand needs new one for every fragment

Primase

synthesize RNA primer, complementary to DNA template

Nuclease

degrade RNA primer, creates gap

DNA polymerase 1 (Repair polymerase)

replaces RNA primer with DNA, use adjacent fragment as primer

DNA ligase

joins DNA fragments together, catalyze phosphodiester bond

DNA helicase

unzip DNA double helix, use energy from ATP hydrolysis

Single strand DNA bonding protein (SSB)

binds exposed single stranded DNA, prevent double helix from reforming, for lagging strand

DNA topoisomerase

Relieves tension in double stranded DNA, make single strandad break in DNA backbone, reseals once tension has been released

Sliding clamp

keep DNA polymerase attached to template

Clamp loader

locks sliding clamp around new DNA double helix, removed and reattached every fragment

Replication of chromosomes end (in eukaryotes)

leading synthesize all the way, lagging strand cannot synthesize all the way, chromosomes become shorter every replication

telomeres

long repetitive nucleotide sequences at end of chromosomes, allow cell to distinguish natural end or random breaks, length varies by cell type and age

telomerase

ezyme, carries own RNA template, add multiple copies of same repetitive DNA sequence, replicates end of eukaryotic chromosomes,remain fully active in rapidly dividing cell

rapidly dividing cells

keep telomerase fully active, cell that line digestive tract and bone marrow cells that generate RBC

Reduced telomerase activity

telomeres eventually disappear and cell cease dividing, hypothesis ofaging, safe guard agaisnt uncontrolled proliferation (cancer)

Types of DNA damage

Depurination, Deamination, Thymine dimers

Depurination

removal of purine (A/G) from a nucleotide, leads to delections

Deamination

cytosine loses amino group, produces uracil, leads to base subsitution (A pair with U instead of G pairing with C)

thymine dimers

UV radiation causes covalent linkages between 2 pyrimidine, creates bulge/distortion in double helix, causes stalling of replication machinery

Xeroderma pigmentosum

inability to repair thymine dimers, high sensitivity to sun, highly susceptible to cancer

Results of unrepaired damage

Base substitution/point mutation, deletion/insertion, Stalling of replication machinery (cannot replicate)

Mechansim of repair

mismatch repair, homologous recombination, nonhomologous end joining

Steps of mismatch repair system

nucleases recognizes damage DNA and removes (cuts sugar-phosphate backbone(excision)) leaving gap in one strand, Repair DNA polymerase (DNA pol I) binds 3’ and fills gap, DNA ligase seals break

mismatch repair system

repairs 90% of errors, removes portion and remakes it correctly, mutation of this are very common in colon cancer

Double strand DNA break causes

mishaps at replication fork, radiation, chemicals

double strand DNA breaks

fragmentation of chromosome, loss of gene, no copies to act as template to reconstruct

Nonhomologous end joining

stick broken ends of dsDNA break back together, specialized group of enzymes (clean up ad ligate), nt lost at site of repair (frameshift mutation)

Homologous recombination

damage to DNA after its been replicated, undamaged double helix acts as template, highly conserved in all cells on earth, no nt lost at repair site

steps of homologour recombination

nuclease degrades 5” end of broken, 3” invades homologous DNA and find complementary sequence (B: recA/ E: rad52), invading is elongated (use undamaged as template), newly elongated joins original partner by base pairing, ligation completes

Mutation

permanent change in DNA sequence (unrepair DNA damage), may or may not alter protein function, natural selection eliminates harmfulones, can remain perserved over tens of millions of years

mutations in germline cells

pass to all cell in multicellular organism (including gametes)

mutation in somatic cells

rise of variant cells, grow and divide in uncontrolled fashion at expense of other cells (Cancer)