Chapter 5 BI203

Activation induced deaminase (AID)

EXPRESSED only in B lymp converts C-->U in DNA removal of U by base excision repair, leaves single strand gap in DNA regulate class switch recombination (CSR) + somatic hypermutation (SHM)

TYPES OF ANTIBODIES arise due to class switch recombination

IgM: activates complement, a first line of defense against invading cells or viruses IgG: activates complement and binds to phagocytic cells and can cross the placenta IgA: antibodies secreted into nasal mucus and saliva to eliminate bacteria/viruses Also in breast milk IgE: against parasitic infections and involved in allergies Allows for antibodies to adapt to pathogens IgM and IgD can switch to IgG,E or A

PROKARYOTE DNA pol EUKARYOTE DNA pol

P= 5 pol genes FOCUS ON I + III E= 15 pol genes pol α, ε, δ

Somatic hypermutation:

Produces mutations within rearranged variable regions of both heavy and light chains --> antibodies w higher affinity These point mutations are directed by uracil:guanine mismatch which will trigger removal and repair

telomeres and telomerase

Cant copy the 3' of lagging strand template Solution - extend old strand. Provides space for primase to create an RNA primer for DNA pol - The terminal sequences = telomeres have tandem repeats of simple seq DNA - Maintained by telomerase Protect chromosomes from degradation (via loop and shelterin) - Simple seq repeats maintain chromosomal length during DNA replication - It's a reverse transcriptase: uses reverse transcription to elongate the existing telomere off of the overhang, and the end of that addition will loop back and interact with the former 3' overhang to create a loop. - Carries its own template DNA - comp to telomere repeat sequences 1. Multiple copies if telomeric sequences can be generated to maintain telomeres 2. Overhanging 3' head of telomeric DNA binds to telomerase DNA --> serves as template for extension of template strand by 1 repeat unit 3. Lagging strand then elongated with RNA priming and DNA pol

Class switch recombination:

Switches between immunoglobulin classes to determine function of antibodies KEEP variable region, change constant region Between highly repetitive sequences in SWITCH (S) region within introns UPSTREAM of each C region - rearranged V(D)J regions combine w diff heavy chain constant regions - heavy chains encoded by a variable region joined to different constant regions

B and T lymphocytes

T LYMP: t cell receptors react w antigens on surfaces T lymphocyte genes formed by site-specific recombination between antibody and T cell receptor genes B LYMP: secrete antibodies that react with antigens

Human telomeres

Telomerase activity maintains telomere length Most cells don't have enough telomerase to maintain telomere for many cell divisions Telomeres shorten as cells age --> cell death Premature aging syndromes due to telomere loss/mutations Cancer cells have high telomerase levels

Sliding clamp proteins and clamp loading proteins (polymerase accessory proteins)

SLIDING CLAMP PROTEINS (PCNA in eukaryotes): load polymerase on the primer and maintain stable association with the DNA template CLAMP LOADING PROTEINS (RFC in eukaryotes): use ATP hydrolysis energy to open sliding clamps and load them onto DNA template clamp-loading and sliding-clamp proteins binds DNA between primer and template. RFC is then released, loading PCNA onto the DNA. DNA polymerase then binds to PCNA. Model of PCNA bound to DNA

Direct damage reversal:

Type of damage addressed: UV induced thymine dimers - altered cyclobutane ring Key molecular players: photolyase by light energy NOT in humans

Translesion synthesis:

Type of damage addressed: bulky DNA lesions that stall replication forks Key molecular players: Bacteria- DNA polymerase V. DNA pol V recognises and targets lesion. replication continues and nucleotide excision repair removes thymine dimer. Eukaryotes- DNA polymerases η, ι, κ, ζ, and Rev1.

Nucleotide excision repair

Type of damage addressed: bulky, helix distorting lesions such as those by UV light induced pyrimidine dimers/interactions with carcinogens Key molecular players: XPC, XPA-XPG, TFIIH complex XPC recognizes disrupted base pairing XPA, RPA, TFIIH bind and unwind (helicase activity) XPG + XPF: endonucleases recruited and cut DNA, excising the fragment/damaged nucleotide

Non homologous end joining:

Type of damage addressed: double stranded breaks Key molecular players: KU70/80 heterodimer, DNA PKcs DNA PKcs - DNA dependent protein kinase catalytic subunit, phosphorylate Ku70.800, Artemis, etc to catalyse these processes Ku70-80 heterodimers: has high affinity for ends of Dna with blunt/sticky overhangs. Binds, then recruits several proteins like ligase Artemis - nuclease that removes nucleotides DNA pol Mu and gamma: add nucleotides

Homology directed repair:

Type of damage addressed: double stranded breaks, during S/G2 phase when a sister chromatid is available as template Key molecular players: BRCA1, BRCA2, RAD51 BRCA1- facilitates resection and loading of RAD51 onto DNA BRCA2- encodes a protein that recruits RAD51 to ssDNA generated at dsDNA breaks

Mismatch repair:

Type of damage addressed: mismatches due to DNA replication errors (adding non comp base) Key molecular players: MutS, MutL, MutH (bacteria) MSH and MLH (humans) MutS recognizes mismatch MutH cleaves the new strand adjacent to the mismatch opposite a methyl group MutS and MutL direct excision between nick and mismatch 3X MORE effective in repairing mismatch on lagging strand --> bc more single stranded breaks on lagging parental strand recognized by methylated adenines after replication.

Base excision repair:

Type of damage addressed: small, non helix distorting base lesions caused by deamination, oxidation or alkylation damage Key molecular players: DNA glycosylases, APE1, DNA polymerase and DNA ligase Eg cytosine --> uracil DNA glycosylase detects base excision Other enzymes removes rest of nucleotide from DNA strand DNA polymerase fills gap and ligase seals it

DNA polymerase role in DNA replication

add a deoxyribonucleoside 5'-triphosphate to the 3' hydroxyl group of a growing DNA chain (the primer strand) Can only extend a pre-existing polynucleotide (primer), which MUST be bound to a template strand by comp base pairing catalyzes the formation of phosphodiester bonds b/w 3' hydroxyl and 5' phosphate

Gene amplification

alters genome structure by increasing the number of copies of a gene - eg rearrangements betw antibody and t cell receptor gene eg in cancer cells (oncogenes) repeated DNA replication makes many copies of a particular --> increase expression. - can occur during development / abnormally in cancer genes RARE - only in specialized cell types

endo vs exonucleases

both cleave phosphodiester bonds endo: cleave within sequence of nucleotides exo: cleave at end of nucleotide sequence + cleave w directionality

homologous recombination

exchange of DNA without altering genes - some rearrangements control gene expression/play evolutionary role --> genetic diversity

T cell receptor

have 2 polypeptide chains = a and b (joined by disulphide bonds) variable and constant regions - genes encoding them are made by recombination between V and J (a chain) or V,D,J segments (b chain)

antibody

have heavy and light region with c terminal constant N terminal variable regions each antibody encoded by unique genes formed by site specific recombination during b lymp development

DNA replication leading and lagging strand mechanisms

leading 5'= continuous lagging 3' = discontinuous - primase makes these short fragments = primers - okazaki fragments (RNA) are added and joined to lagging RNA polymerase synthesise fragments de-novo (w/o primer) RNA primers removed by exonuclease activity + gap filled by DNA polymerase DNA ligase joins DNA fragments in prokaryotes: DNA pol I does both in eukaryotes: RNase H removes RNA primers (degrades the RNA strand by 5'-3' exonuclease) and DNA pol δ fills gaps

Origin of replication (ori)

replication of genomic DNA is initiated here In e.coli = 245bp DNA sequence In e.coli, initiator protein (DnaA) binds to ori Unwinds DNA + recruits other proteins for DNA synthesis (DNAB HELICASE COMPLEXES) Helicase and ssDNA binding proteins cont unwinding and exposing DNA template + primase initiates synthesis --> 2 replication forks formed and move in opp directions along circular chromosome In eukaryotes, multiple origins of rep are needed to replicate larger genome 30000 origins Prod 2 replication forks

gene amplification in amphibians eggs and drospholia

ribosomal RNA genes amplified to around 1mil copies --> prod proteins required for early development in drosophila: genes that encode egg shell proteins are amplified in ovarian egg cells

Topoisomerases

"Supercoiling" of parental DNA ahead of the replication fork must be relieved As template DNA strands unwind, DNA ahead replication fork is forced to rotate in the opposite direction --> causing circular molecules to become twisted around themselves Topoisomerases catalyze the reversible breakage and rejoining of DNA strands. The transient breaks by this serve as swivels --> 2 strands of DNA to rotate freely around each other. Topoisomerase II break and rejoin double-strand DNA molecules. Inhibiting topoisomerase II would be disruptive to DNA replication and the separation of intertwined DNA molecules during mitosis.

Proof reading by DNA polymerase

- G incorporated instead of A. 3' terminal G is not H bonded to template strand - polymerase recognizes mismatch - polymerase uses 3'-5' exonuclease activity to remove incorrect nucleotide need for proof reading --> evolutionary pressure: 1. DNa pol dependence on annealed primer 2. 5'-3' directionality of synthesis

helicase + SSB roles

- helicase catalyses the unwinding of DNA (needs ATP) - parental dsDNA needs to be separated and stabilised as SSDNA -- CMG Helicase complex migrates along the leading strand template as it unwinds the double strand. SINGLE STRANDED DNA binding proteins (SSB) stabilise unwound DNA, keeping it single stranded so that it can be copied by DNA polymerase

dna damage examples

1. deamination of cytosine - C --> U. blocks transcription/replication 2. exposure to UV light: uv light induces formation of pyrimidine dimers --> 2 adjacent pyrimidines are joined by a cyclobutane structure --> saturation of double bonds between carbon 5+6 3. reaction w carcinogens. react w DNA adds large chemical groups to DNA

An S.Cerevisiae ARS element

An S.Cerevisiae ARS element has 11bp consensus sequence + other elements (B1, B2, B3) Origin recognition complex (ORC) binds to ACS and B1. ORC recruits more proteins (2 hexamers of MCM27 DNA helicase) to origin Binding of GINS to each mcm hexamer --> forms active helicases that unwind DNA --> replication forks that move in opp directions ORC dissociates from ori Ori's are much less well defined in other eukaryotes - may be defined by chromatin structure

Antibody diversity: Site specific recombination

Mediated by RAG recombinase enzymes LIGHT CHAIN Recombination between V and J --> 600 possibilities HEAVY CHAIN: 2 recombination's. D with J & V with DJ. --> 7200 possibilities

RAG PROTEINS recombinase enzymes

mediate V(D)J recombination immunoglobulin and t cell receptor genes (V+D) are flanked by recombination signal (RS) sequences = in opp orientations at end of 5' and 3' seqs - RS seqs recognized by a complex of RAG 1+2 proteins cleave DNA between coding and RS sequence - broken coding strands --> joined --> gene segment - mutations from loss of bases at end of NHEJ/addition of bases - alter sequences --> end up with diff antibodies