DNA Lecture 7-9

principles of DNA replication

parental strand is used as a template where the daughter strand is complementary, the phosphate group in the incoming deoxynucleotide triphosphate reacts with the 3’

hydroxyl group in the growing DNA chain, the opening of the DNA duplex

occurs progressively in cells forming the DNA replication fork

replisome

protein machinery that operates at the replication fork

helicase

unwinds duplex DNA

CMG helicase and Replication Protein A

work together where CMG helicase is a specific, multi-subunit eukaryotic replicative helicase complex that generates single-stranded DNA (ssDNA) and Replication Protein A coats the exposed ssDNA templates

topoisomerase

removes supercoiling by transiently cutting DNA

dNTP

energy source used by cells to synthesize DNA with four dNTPs matching the four DNA bases (N=A,C,T,G)

orientation of DNA replication

leading strand is 3’ to 5’ (so the daughter strand can be 5’ to 3’) and the lagging strand is 5’ to 3’(leading to okazaki fragments)

primase

synthesizes a short RNA primer complementary to the DNA template because DNA polymerase requires an existing 3'-OH end to attach new nucleotide first

DNA polymerase α (Pol α)

extends the primer to eventually form a new daughter duplex DNA

Okazaki fragments

short discontinuous fragments consisting of RNA and DNA on the lagging strand

RNA primer

a short, single-stranded sequence of ribonucleotides that acts as the required starting point for DNA replication

DNA polymerase δ (Pol δ)

takes over from Pol α to synthesize the lagging strand

DNA polymerase ε (Pol ε)

takes over from Pol α to synthesize the leading strand

DNA ligase

seals nicks and connects Okazaki fragments by catalyzing the formation of a phosphodiester bond between a 5′ phosphate and a 3′ hydroxyl group

PCNA (Proliferating Cell Nuclear Antigen)

a ring-shaped protein that acts as a sliding clamp around double-stranded DNA to prevent Pol ε and Pol δ from disassociating from the template

RFC (Replication Factor C)

couples ATP binding and hydrolysis to open and close the PCNA ring around primed DNA

RFC/PCNA complex

RFC and PCNA work together to load ring-shaped protein clamps onto DNA during replication to prevent the DNA polymerases from sliding off the template strand

Ribonuclease H and FEN-1

displace the RNA component at the 5’ ends of the Okazaki fragment

replication origins

initial unwinding of DNA that tend to be AT-rich throughout all the DNA (not just one starting point)

Origin Recognition Complex (ORC)

six subunit protein that mark replication origins and load helicase

MCM helicase

specialized helicase that is loaded during G1 but activated in S through phosphorylation and protein interactions

Cyclin-dependent kinases (CDKs)

enzymes that regulate the cell cycle, transcription, DNA repair, and metabolism

directionality of replication

bidirectionally

DNA Polymerase α–Primase

complex that synthesizes an RNA primer and extends it with a short DNA tail

hydrolytic depurination

the spontaneous chemical breakdown of the glycosidic bond between a purine base A or G and a deoxyribose sugar in DNA (2,000 – 10,000 per day per cell)

cytosine deamination

a chemical reaction that transforms cytosine into uracil (once every 5 days per cell)

methylation

a methyl group is added to a DNA strand (600 times per day per cell)

mutations

permanent and transmissible changes to the genetic material of a cell that can

occur spontaneously, by transposable elements, and by errors during replication

mutagens

chemical compounds, UV radiation or ionizing radiation that increase the frequency of mutation

carcinogen

agent that causes cancer and many are mutagens

proof reading activity

DNA polymerases δ and ε but not α have a 3’ to 5’ exonuclease or “proofreading” activity

Basics of DNA repair

detection and removal of the affected region

Mismatch Repair (MMR)

a cellular system that identifies and fixes wrong nucleotides and works close to the replisome to quickly fix mistakes

MMR sequence

MSH2 and MSH6 bind to daughter strand, MLH1 endonuclease makes cut of DNA, helicase unwinds, DNA exonuclease digests segment of daughter strand, gap is repaired with Pol δ and DNA ligase

Base Excision Repair (BER)

a cellular mechanism that fixes small, non-helix-distorting DNA base lesions (like cytosine spontaneously transforming into uracil)

BER sequence

DNA glycosylase hydrolyzes the bond between the mispaired base and the sugar-

phosphate backbone, Apurinic/apyrimidinic endonuclease 1 (APE1) cuts the DNA backbone, AP lyase removes the deoxyribose phosphate, gap is repaired with Pol δ and DNA ligase

Nucleotide Excision Repair (NER)

DNA repair pathway that removes bulky, helix-distorting lesions like two wrong nucleotides

NER sequence

proteins 23B and XP-C do the initial recognition of the DNA lesion, TFIIH, RPA and XP-G catalyzes the unwinding of the DNA region around the lesion, XP-F and XP-G generate cuts 24-32 bases apart, releasing the fragment carrying the lesion which will be degraded, gap is repaired with Pol δ and DNA ligase

how DNA repair and replication have complications

during DNA replication, the replisome may encounter lesions on DNA that were

not previously repaired and DNA polymerases will stop preventing replication

translesion (TLS) polymerases

a special type of DNA polymerase that are able to bypass a lesion, using the damaged DNA as a template but often insert the incorrect base and are error prone

single and double stranded DNA breaks

single stranded breaks are common and efficiently repaired with DNA ligase but double stranded breaks also occur during DNA replication which is harder to repair

homologous recombination (HR)

uses a sister chromatid or homologous chromosome as an exact template to restore the original sequence accurately

HR sequence

MRN degrade the damaged DNA ends leaving an overhang on each strand of 3-4 nucleotides, the sections are stabilized by a RPA proteins until RAD51 binds to the DNA forming a filament, the filament binds to a sister chromatid, the invading strand searches for matching base pairs (strand invasion), if a sequence matches for at least 5 triplets DNA polymerase uses the invading strand as a template, then the second complementary strand gets used as a template and DNA polymerase does its thing, then DNA ligase cleans it up

why double stranded may be produced

during meiosis to increase genetic variability

Non-Homologous End Joining (NHEJ)

repairs double stranded breaks in DNA by directly ligating the broken ends together

types of DNA repair

Mismatching Repair (MMR), Base Excision Repair (BER), Nucleotide Excision Repair (NER)

types of double stranded break repairs

homologous recombination (HR) and Non-Homologous End Joining (NHEJ)

NHEJ sequence

A complex of DNA-PK and Ku80/Ku70 binds to the ends of the break, ends are digested by nucleases or filled in by DNA polymerases, ends are ligated together

why NHEJ is error and chromosomal translocations prone

because there is no template and ends are simply ligased back together, it results in a loss of information, either by the removal or the addition of nucleotides

chromosomal translocations

a type of genetic mutation where a piece of a chromosome breaks off and attaches to another homologous chromosome

when NHEJ or HR repair is used

NHEJ is used during G1 phase since only one copy of the genome is present, HR is used during S, G2, and M phases

BRCA1 and BRCA2 in HR

mutations that inhibit the function of these proteins are associated with a predisposed development of cancer, particularly breast and ovarian

PARP inhibitor

a class of targeted oral cancer medications that make sure cancer cell double strand breaks cannot use HR and therefor commit suicide

why chemotherapy works

it targets cancer DNA which isn’t easily fixed