DNA Replication, Gene Expression & Protein Synthesis Flashcards
DNA structure
3D structure, 2 helices wrapping around each other, surface as major (wide spiral) and minor grooves (narrow spiral), size of groove affects access to bases within helix
DNA and drug pharmacology
target for chemotherapy drugs through various types of interactions, strand breaker, non-covalent interactions: not chemically interacting but fitting, covalent complexes: cis platin - platinum-based
Non-covalent interactions with DNA
intercalation between bases - fitting into (in space/opening), minor grooves, major grooves, or spanning both
Cis platin
anticancer chemotherapy drug, covalent binding - specific to 1 strand (nucleotide-nucleotide or to protein), involving at least one guanine, multiple ways of binding increase effectiveness as drug
Types of covalent cis platin binding
always to 1 guanine nucleotide, interstrand - consecutive nucleotides opposite strand = about 3-5% (one strand across another), intrastrand - consecutive nucleotides, about 80-90% (2 covalent interactions on one strand - same backbone), intrastrand - nearby nucleotides, about 3-5% (skip residue), either strand - guanine and protein, about 3%
How and why is cis-platin binding detrimental to cancer cell? are only cancer cells affected?
covalent bonds across helix or along one strand block RNA or DNA polymerase decreasing DNA replication or mRNA production for cell growth, DNA is template for polymerase, slower growing normal cells less likely affected than faster replicating/metabolizing cancer cells, chemotherapy has a negative impact on cancer cells, nonselective so not only cancer cells impacted (all cells that replicate/transcribe DNA)
DNA replication overview
replication events/requirements, chromosome ends (5’ to 3’ rule) and unwinding (topoisomerase), possible targets for chemotherapy, topoisomerase unwinds double strand DNA, 5’ to 3’ rule because added onto exposed 3’ hydroxyl, double helix slows down DNA polymerase so needs to be unwound by topoisomerase
the 5’ to 3’ rule
templates are anti-parallel, RNA primer required to provide initial 3’ OH for DNA nucleotides added on replication proceeds 5’ to 3’, replication fork moves, RNA primer (short stretch of RNA) binds antiparallel and daughter strand synthesis starts after on 3’ OH available for DNA polymerase
2 kinds of lagging strands
leading strand - continuous synthesis (faster), one RNA primer and synthesis towards replication fork: lagging strand - short and discontinuous synthesis yields separate lengths of DNA (Okazaki fragments) so slower, RNA primers removed by RNAseH enzyme, DNA nucleotides fill in gaps, fragments joined by ligase, several priming events (slower), use 3’ end of RNA for DNA
Ligase
ligase completed, RNA primers come off template before they are incorporated, covalently ligated together for one covalent strand, before they are not covalently linked, when RNA primer removed, 3’ OH available to fill
A consequence of the 5’ to 3’ rule
leading and lagging strand, chromosome ends (telomeres) shorten with repeated round of DNA replication, single-stranded DNA template at chromosome terminus left after removal of RNA primer is degraded by exonucleases, gaps remain unfilled and single-strand is degraded, single stranded has no replication
What are the consequences of repeated rounds of DNA replication?
shortened each time cell replicates DNA, both ends of chromosome (telomere) are shortened
Chromosome ends
telomeres = ends of linear eukaryotic chromosomes, protect and stabilize internal section of chromosome, conserved, short, highly repeated sequence, about 6-9 nucleotide (similar but can vary), keep an eye on that TTA series, do not contain any protein coding but if degraded too much - start to degrade protein coding region
Telomere shortened too much
cell stops replacing (loses ability to replicate DNA), sign of aging in cells, single stranded region degraded (telomere shortened), telomere shortening may = ‘biological clock’ of cellular age, no mitosis but can still transcribe, etc.
‘End replication problems’ of DNA synthesis
DNA polymerases need RNA primer to start replication, 20 to 200 single strand end left after removal of terminal primer, single strand region removed by exonuclease and chromosome shortens
How to rebuild telomeres
cell can slow progressive degradation if right enzymatic activity, telomerase = catalyzes telomere lengthening (enzyme for mitosis forever), functions as reverse transcriptase synthesizing DNA from RNA template - contrast to typical DNA replication, enzyme function requires protein and RNA subunit, adds nucleotides to single-stranded overhang which becomes long enough to pair with usual RNA primer, single-stranded portion now long enough to be primed by usual means, long enough for daughter strand (but some shortening), balance between telomerase enzyme and DNA replication
Telomerase structure
RNA template (with uracil) in backbone and associated protein, adds DNA onto 3’ hydroxyl, temporarily/transiently base pairs, template within (RNA) should extend strand - complementary to repeats making telomere
Telomerase in normal cells
majority of normal cells do not produce telomerase (somatic cells), consequences for tissue/organ, telomeres shorten then cell stops dividing then replicative senescence, accumulated tissue “wear and tear” without new cell replacements, if re-extension of telomeres then continued cell replication, cellular aging/dying
Telomerase activity
reproductive cells: moderate levels, blood, skin, and gastrointestinal cells: very low levels, tissues where replacement and renewal is critical, some shortening still crus because level is too low, crypt is location for high replication (more telomerase) and lower levels of telomerase, at the time the cells are shed and replaced by replicative cells at the crypt (travel up), skin cells have a low level of replacement = low telomerase
Cancer cells and telomerase
about 90% of cancer types have high telomerase levels, telomerase levels increases from early to late stage cancer, telomeres maintained and cells divide and escape replicative senescence (no internal clock), the other about 10% of cancer cells maintain telomere ends, alternative lengthening of telomeres = sister chromosome serves as template in process similar to homologous recombination, extended telomere of matched chromosome
DNA replication as drug target
telomerase function blocked by “antisense” DNA, telomerase function blocked by AZT
Telomerase function blocked by “antisense” DNA
restore clock to stop replication, complementary to DNA component of enzyme (plug up to preoccupied with something else), inactive telomerase complex (DNA+RNA+protein), RNA not available as template for telomere extension, synthesize short bit of DNA that base pairs and blocks telomerase activity with RNA
Imetelstat
administered by IV infusion, green “tail” is 16 carbon-long lipid to improve movement across cell membrane to increase potency and improve pharmacokinetic and pharmacodynamic properties (covalent binding), membrane is compartment barrier (to nucleus), imetelstat binds to template region of RNA component of telomerase, resulting in direct, competitive inhibition of telomerase enzyme activity (into nucleus), clinical trials results: suppresses proliferation of malignant progenitor cells aiding recovery of normal hematopoiesis in patient with hematologic myeloid malignancies
Telomerase function blocked by AZT
azidothymidine: structure does not allow additional nucleotide to be added which is consequence of 5’ to 3’ rule, at end of chromosome, no more 3’ OH because needed so terminated
Thymidine vs. zidovudine (AZT)
different chemical structure, thymidine - binds to previous nucleotide (3’ OH group allows binding to next nucleotide), continues nucleic acid chain, needed in telomere; AZT - binds previous nucleotides, azido (-N3) group in 3’ position (not available for 3’ elongation), thymidine analogue, phosphate group of next nucleotide cannot binds azido-thymidine; synthesis stops
Topoisomerases
break and restore backbone, nuclear enzymes supporting DNA replication, dual functionality with covalently binding to DNA, induce single-stranded breaks (covalent) then DNA unwinds helix is relaxed so ssDNA available for replication (DNA polymerase), topoisomerase rejoins DNA ends in relaxed region dissociates from relaxed double-stranded region, double helix not conductive to polymerization
Topo
elevation, major and minor DNA, impede DNA replication with in helix
What happens if religation is inhibited?
accumulation of single-stranded breaks, irreversible double-stranded breaks, leads to cell death
Drug helix as drug target
Camptothecins = natural and synthetic alkaloids (cytotoxic chemotherapy drug) - insert between DNA base pairs (intercalation because planar), partially inhibit topoisomerase 1, initial cleavage occurs, drug hydrogen bonds to topoisomerase and intercalates between base pairs, religation step is inhibited, single-stranded breaks accumulate (to double strand breaks)
Clinical use of camptothecin
topoisomerase activity required at beginning of S phase to relax helix and allow access for DNA polymerase, cancer cells must be in S phase (most activity), little effect with slow or non cycling cells, treatment duration depends on cell cycle length, only active when cells replicate DNA
Chromatin - clinical therapies
chromatin = DNA and associated proteins, entinostat = specific inhibitor of class 1 HDACs, HDACs = histone deacetylases, trials histone enzymes inhibitors
Phase 1 clinical trials
test a new drug in a small group of people (10-80) for the first time to evaluate a safe dosage range and side effects
Phase 2 clinical trials
drug or treatment is given to a larger groups of people (100-300) to see if it is effective and to further evaluate its safety, HDACs modify chromatin protein
Phase 3 clinical trials
drug is given to large groups (1,000-3,000) to confirm effectiveness, monitor side effects, compare to commonly used treatments, and collect information on safety, HDACi at all phases
Phase 4 clinical trials
post trail studies for additional information including drug’s risks, benefits, and optimal use, histones enzymes inhibitors
Gene expression and regulation
DNA to and from protein interaction affects gene expression (post transitional modifications of chromatin protein, histones, control transcriptional access to gene), genome (DNA sequence) and epigenome (chemical modification to DNA and associated proteins) to phenotypic traits, coding information from DNA to mRNA, nuclear import/export issues for modifying enzymes and mRNA
Coding information from DNA to mRNA
transcription initiated at promoter region, endogenous and clinically used compounds (like certain drugs and hormone s) can affect access to promoter and therefore gene expression (transcription)
Sequence of gene expression and regulation
DNA sequence to mRNA to protein to cell, non-sequence modification to DNA and chromatin protein also influence expression (histone acetylation)
Chromatin structure DNA and protein
histones: small (about 120 amino acids) and basic proteins, contain numerous lysines, lysine R group has positively charged DNA phosphate, 5 histone types which are 4 types in core and 1 as spacer between nucleosomes; nucleosomes: 4 pairs of histones at core, about 146 base pairs DNA wrap around in about 1.7 turns
