Repetitive DNA Sequences & DNA Profiling Flashcards
Genome
the total set of DNA in an organism
Highly repetitive DNA
Satellite DNA
Satellite DNA
-constitutes ~5% of the human genome
-consists of relatively short sequences repeated a large number of times
-present as tandem repeats clustered in specific chromosomal areas
-typically heterochromatic and located within or near centromeres
*a notable sequence in humans is the alphoid family
~170 bp in length present in tandem arrays of up to 1 million bp’s
not transcribed – play an undefined structural role
Middle Repetitive DNA
Tandem Repeats
Tandem Repeats
-variable number tandem repeats (VNTRs)
minisatellites
15-100 bp sequences interspersed w/in and b/w genes
-short tandem repeats (STRs)
microsatellites
2-5 bp sequences
-multiple-copy genes (e.g. rDNA)
Transposable Elements
-“jumping genes”
-can move (transpose) w/in and b/w chromosomes
-may be either …
*retrotransposons – jump by using an RNA intermediate & subsequent reverse transcription
*DNA transoposons – move in and out of the genome as DNA elements only
-abundant in many organisms from bacteria to humans
Inverted terminal repeats (ITRs)
-located on each of the transposable element
-9-40 bps long; identical in sequence but inverted relative to each other
Open reading frame (ORF)
encodes for the enzyme transposase
Short direct repeats (DRs)
flank each transposable element insertion
Autonomous DNA transposons
-encode their own functional transposase
-have intact ITRs
-able to transpose by themselves
Nonautonomous DNA Transposons
-do not encode their own functional transposase enzyme
-cannot move on their own
-require presence of an autonomous transposon somewhere else in the genome
*will use its transposase enzyme
How do transposons move?
-discovered initially by Barbara McClintock in the late 1940s
-usually, transposons move by “cut-and-paste” mechanisms
*original site containing the transposon is typically repaired accurately
Retrotransposons
-make up ~42% of the human genome
*compared to DNA transposons, which make up ~3%
-may also be either autonomous or nonautonomous
-like DNA transposons, they encode the proteins that are required for their transposition & flanked by direct repeats
Structural Elements Retrotransposons
-ORFs encode two enzymes:
reverse transcriptase
integrase
-the ORFs are flanked by long terminal repeats (LTRs)
*contain the promoter and polyadenylation sites for the protein-encoding ORFs
*LTRs are absent in some retrotransposons
SINES (short interspersed elements)
100-500bps long; may be repeated up to 500,000x
13% of genome
example: the Alu family of DNA sequences
200-300 bps long dispersed uniformly throughout the genome
this family alone encompass >5% of our whole genome
some are transcribed but have unknown role
Middle Repetitive DNA
Interspersed Transposons (SINES and LINES)
LINES (long interspersed elements)
1-6kb in length; repeated ~850,000x in the human genome
21% of genome
example: the L1 family of DNA sequences
Transposable Elements in Disease
-TE’s can cause mutations if they “jump” to certain places
inserted into a gene’s coding region (frameshift)
inserted into a regulatory site
promoter, enhancer, silencer
polyadenylation sequence, splice site
-known to be linked to cases of …
hemophilia
muscular dystrophy
neurofibromatosis
familial breast cancer
acholinesterasemia
Proofreading
-DNA polymerases have 3’ to 5’ exonuclease activity
-cuts out the incorrect nucleotide (from the end of the DNA that it just added it to), and replaces it with the correct nucleotide
-DNA polymerase’s error rate is ~ 1 in 100,000 bases (10-5)
*proofreading catches 99% of those errors, improving efficiency ~100 fold for a final error rate of 10-7
Mismatch Repair
-incorrect nucleotide is removed & correct one is inserted in its place
-carried out by suite of MMR proteins:
endonucleases nick the phosphodiester backbone
exonucleases unwind & degrade the nicked strand
polymerase fills in the gap
ligase seals the newly synthesized piece to rest of strand
Clinical Connection to DNA repair
-hereditary nonpolyposis colon cancer
*Lynch Syndrome
-mutations in genes that encode for MMR proteins
*less able to repair mutations
*mutations begin to accumulate
*increases risk for cells to become cancerous
-affects many other tissue types besides colon
Base Excision Repair
-repairs damage to individual bases on a single DNA strand
*alkylation or other chemical modifications
3 Basic Steps to Base Excision Repair
- DNA glycosylases cut the glycosidic bond b/w target base and its sugar
- AP* endonuclease cuts the phosphodiester backbone
- DNA polymerase and ligase fill in the gap & seal it
Nucleotide-Excision Repair
-removes bulky lesions
*e.g. pyrimidine dimers or bulky adducts that distort the DNA helix
-similar to BER, but excise an oligonucleotide instead of 1 base
-multiple proteins involved
*Encoded by >30 genes in humans!
Clinical Connection to DNA repair 2
xeroderma pigmentosum
rare recessive disorder
predisposes affected individuals to severe skin abnormalities, skin cancers, & other developmental / neurological defects
Double Strand Breaks
-occur when both strands of the DNA helix are damaged, not just one
-worst form of DNA damage
*break chromosomes apart
*create unprotected ends of the DNA if not corrected, the DNA gets degraded
-caused by free radicals and ionizing radiation
-repaired by one of two major mechanisms
Homologous Recombination Repair (HRR)
-DS break recognized & degraded, leaving 3’ overhangs
-the 3’ overhang invades the homologous DNA duplex on the sister chromatid
-complementary sequences align & DNA polymerase uses the homologous sequence to extend the 3’ ends
-heteroduplex gets resolved
Non-Homologous End Joining (NHEJ)
-does not recruit a homologous region of DNA
-activated during G1 (before DNA replication)
-a complex of many proteins bind to the free ends of broken DNA
*trim the ends
*ligate them together
-highly error-prone & may lead to abnormal chromosomal translocations
