DNA Replication and Repair Flashcards
anti miRNA
singled stranded, chemicaly modified ASO bind to miRNA to block activity. miR-122 miRNA is therapy for HepC virus.
Components of a NT
nitrogenenous base attached on 1’ of ribose ring ribose phosphate group attached to 5’ carbon of ribose
Base Excision Repair
repairs those missed by NER, and that do not distort DNA
1) modified base recognized by DNA glysoylase which hydrlyzes glycosidic bond (base and sugar) to yield a-basic site.
2) AP site specific endonuclease cleaves Sugar phosphate backboke from 5’ to AP site
3) a different endonuclease cleaves Sp backbone from 3’ to AP site
4) DNA pol fills in gap and nick is sealed by ligase
Methylation of Adenine
this is done in bacterial cells and is used as an identifyer for DNA older strands
how are small RNAs delivered?
1) put them inside lipid nanoparticle with an external lipid layer that targets a particular region, where it is then take up in a lysocome and ssRNA comes in contact with mRNA 2) chemically modified so they do’t get degraded and have glycol-conjugate that is specific 3) virus expresses gene and makes RNA that folds back on itself.
how do Streomyces that make puromycin protect themselves?
they also make an enzyme that inactivates antibiotic in the bacteria itself. modifies the part of puromycin that is involved in the covalent attachment of polypeptide chain, rendering it inactive.
DNA Pol I vs DNA Pol III
1: distributive - dissociates easily replaces RNA primers Exonuclease 5’ to 3’ 3: processive both have Pol 5’ to 3’ and exonuclease 3’ to 5’
Cockayne syndrome
super hypersensitivity, premature aging, mutation in transcription NER
RNAi
endogenous gene silencing mechanism by siRNA and miRNA ds RNA induce translational inhibition or degradation of mRNA containing partially complimentary sequences.
When does Loss of Heterozygosity Occur?
Mis-regulation of HR use of other chromosomal homolog as template instead of sister chromatid
Nucleotide Excision Repair
Removes lesions that distort DNA structure \ block DNA and RNA pol movement ex: thymine dimers, DNA adducts Recognition of damage is by multi-protein complex, but all other steps are the same as BER
Rag Recombinase
initiates DS breaks in NHEJ to create antibody diversity
Steps NHEJ with Ku
1) Ku recognizes ds break and binds to protect form exonuclease 2) Ku recruits DNA-PKcs which autophosphorylates to attract ARTEMIS to remove DNA damage 3) polymerase fills in gap 4) Ligase 4 seals
Camptothecin
chemo drug targets topoisomerase
Dicer
ribonuclease of RNAse III that cleaves dsRNA into miRNA
How are DS breaks sensed?
PIKKs 1) ATM and ATR
How does BRCA1 promote HR?
phosphorylated by ATM and ATR to recruit to DS break. Depends on activated by CDK, when present, this pushes towards HR.
Dideoxyinosine
DDI inhibits DNA synthesis by the reverse transcriptase enzyme HIV medication Chain terminal mimic of nucleotide
What methods are for direct reversal of Damaged bases?
1) ssDNA break reversal by ligase
2) reversal of UV damage by photolyase
3) reversal of base alkylation (O6 methyltransferase MGMT)
what happens with imperfect match of miRNA?
transcriptional repression
Tat
The first protein synthesized in the human genome of HIV that leads to further expression of the viral genome. There is a tat mimic that fits nicely into the groove of HIV-TAR (tat activated region) to prevent replication of HIV
BER vs NER
BER targets damage that does not distort DNA NER targets damage that does distort DNA
BER: recognized by specific glycosylase
NER: recognized by multiprotein complex
Holliday Junction Resolution
can make two cuts Vertically: recombinants (DSBR) Horizontally: no recombinatns (synthesis dependent strand anneals)
Intercalation drugs
Actinomycin D
Doxorubicin
MGMT
is a direct reversal type of DNA repair that reverses base alkylation
removes added methyl group and adds it on itself Glioblatomas have MGMT silenced
How can RNAi be made in body?
make small RNA in vivo, purify the enzyme and let is up the RNA introduce it to cells to cleave mRNA in cell. Introduce gene that will express RNA that will naturally fold back on itself to generate siRNA or miRNA that will specifically target domain for degradation.
Cisplatin
alkylating agent drug that reacts with nitrogen in the bases to cause crosslinking. This causes too much damage in the cellular DNA when cells are dividing very rapidly and signals them to be sent into apoptosis. Chemotherapy drug.
Xeroderma Pigmentosum (XP)
sun hypersensitivity, pigmentation disorders mutation in Global Genome NER
Base Alkylation
Addition of hydrocarbon groups on nitrogenous base to form large covalent adducts to DNA that block DNA replication and transcription machinerly Benzenes and derivatives of coal and cigarette smoke
De-Purination
low pH promotes b-glycosyl bond between base and sugar makes phosphate backbone sensitive to breakage
siRNA
22 nt generated from longer doubled stranded RNA precursors by ribonuclease dicer Immunity to virus mRNA degradation influences; Chromatin formation, transcriptional silencing, mobile element repression binds in perfect match
Actinomycin D
naturally occurring antibiotic used in chemotherapy
Cyclic peptides that interact with DNA in a non-covalent manner. Stacks with DNA bases to alter double helical structure in intercalation.
Interferes with DNA replication and transcription.
DNA Polymerase I
a distributive enzyme Replaces RNA polymerase three functions: 1) DNA polymerase 5’ to 3’ 2) exonuclease 3’ to 5’ (proofreading) 3) exonulcease 5’ to 3’ (primer removal)
DNA Pol III
Processive responsible for replicating DNA lagging and leading strands Holoenzyme made for 17 proteins DNA pol 5’ to 3’ Exonuclease activity 3’ to 5’
what happens with perfect match of miRNA?
cleavage and destruction of mRNA
Methylation of cytosine
5’ position methylated. does not affect base pairing majorly, but represses transcription of DNA sequence. Only occurs at C’s with an adjacent 3’ G.Thus it is thought to be symmetrical
DNA damage checkpoint
Stalled replication fork, DNA damage, ds break is detected by ATR and ATM, which recruit active signaling Chk1 an Chk2 leads to production of DNA repair proteins reversible cell cycle arrest permanent cell cycle arrest
What determines HR vs NHEJ?
1) tumor supressor presence a) BRCA1 (HR) or 53BP1 (NHEJ) 2) Point in pathway a) HR is dependent on being in G2 of S 3) protection of ends a) nonprotected (HR) b) protected (NHJE)
Exon Skipping
target intra-exon junctions or splicing regulatory elements binding to the site inhibits splicing and forces the choice of alternative splice sites.
How does extreme pH influence Tm?
alter ionization of groups on bases that participate in H bonding
Micro RNA
21 NT translation repression and degradation
Drosha leaves miRNA to be exported to cyto
Dicer binds to miRNA in cyto and then binds to Argo complex to form Argo-Risk complex
what disease have mutations in tRNA?
myloclonic epilepsy ragged red riber disease
What makes DNA Pol III processive?
Proliferating Cell Nuclear Antigen (PCNA) wiht the beta-clamp ring
Artemis
works in NHEJ attracted by Ku and DNA-PKcs both endonuclease and exonuclease to be able to remove damaged DNA caused by ds break
how does miRNA target mRNA?
base pairing of miRNA to target mRNA to 3’ UTR serves to guide effector complex Seed region is critical a single miRNA will have multiple binding sites in one 3’ UTR or a single 3’ URT will be attached by different miRNA
Purine
two cyclic nitrogenous base Adenine Guanine
Spo11
programs DS breaks in HR leaves DS breaks with overhang mutation in ATM leads to more initiation of Spo11
Seed Region
nucleotides 2-8 form the seed region and determine whether miRNA will target the region
Doxorubicin
intercalator
miRNA biogenesis
miRNA folds on itself to generate ds RNA structure
Spontaneous Deamination
Changes a cytosine to a Uracil in DNA Repaired by Base exision repair 100-500 per cell per day
Puromycin
necleotide analog that mimics tRNA to terminate translocation covalently links to amino acid
Glycosylase in BER
flips out damaged base and removes by hydrolyzing bond specific to particular base damage, inappropriate base or mispairing
NHEJ
imperfect, loss of few NTs, occurring throughout cell cycle most associated with Immune function and antibody diveristy of VDJ either through Rag pathway or Ku with DNA-PKcs wuick repair, not sequence dependent
RNA-Induced Silencing Complex
catalytic effector complex of RNAi mediated gene silencing. multiprotein complex that works with arg to incorporate one strand of siRNA and miRNA.
Myloclonic Epilepsy and Ragged Red fiber disease
mutation in tRNA gene
Arognaut
bind to small RNAs and are conserved in all domains. mediate target recognition. multiple argonauts in cell, depending on what arg binds to small RNA, function will dependentant
Action of 53BP1
positively regulates NHEJ by promoting synapsis of distal broken ends blocks resection of 3’ OH end
How is mismatch repair different in P vs E?
MutS and MutL in bacteria recognize damage
MSH and MLH in mammals
Bacteria - old strand is methylated
Euk: new strand has more nicks due to the okazaki fragments and RNase H2.
Mismatch NT repair
during replication and before methylation
MutS and MutL (B) and MSH and MLH (E) recognizes DNA errors and Pol III cleaves bond, exonuclease chew away strand with mismatch while helicase unwinds, Pol Adds dNTPs to gap and ligase seals
Lynch Syndrome
Due to Mismatched Base Repair mutation in humans Hereditary Non-Polyposis Colorectal Cancer increased risk of colon cancer
drugs that target topoisomerase
Etoposide and camptothecin
RNase H-dependent ASO
binds to complimentary mRNA and cleaves site to reduce gene expression or block ribosome.
Homologous Recombination (basic)
Perfect repair, requires sister chromatids, limited to S and G2 no gain of loss of NTs assures genetic diversity of gamets Can act with BRCA1 to form holliday junction
how does NER recognize damage?
Global Genome NER: recognizes distortions in whole genome Transcription coupled NER: recognizes damage in actively transcribed regions
De-Amination
cleavage of amine group, leading cytosine to be transformed into thymine. damage is hard to detect sometimes naturally, but caused by nitrous acid in smoke and preservatives
piRNA
24-27 NT deviation can vary in different organisms and loci, flies and mammals form long precursors and worms form short. RNA protein complex that interact with piwi proteins. Function: immunogenesis maintenance of germ line controls of mobile elements (transposons)
Stability of components of nucleotides
Pyrimidine is more soluble than purine. Nucleotide > nucleoside > base
VDJ Recomination
each heavy and light chain contains variable repeats Constant, variable, Diversity (H) and Joining VDJ is recombination so that there is one copy of each segment in a given lymphocyte leads to enormous antibody repertoire.
Drosha
RNAse III that is implicated in processing newly transcribed primary miRNA in nuclease.
Base Alkylation
Addition of methyl group to cause point mutation ex. BP: CG becomes GT and eventually mutates to AT
