Repetitive DNA Sequences & DNA Profiling

Question 1

Genome

Answer 1

the total set of DNA in an organism

Question 2

Highly repetitive DNA

Answer 2

Satellite DNA

Question 3

Satellite DNA

Answer 3

-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

Question 4

Middle Repetitive DNA

Answer 4

Tandem Repeats

Question 5

Tandem Repeats

Answer 5

-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)

Question 6

Transposable Elements

Answer 6

-“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

Question 7

Inverted terminal repeats (ITRs)

Answer 7

-located on each of the transposable element
-9-40 bps long; identical in sequence but inverted relative to each other

Question 8

Open reading frame (ORF)

Answer 8

encodes for the enzyme transposase

Question 9

Short direct repeats (DRs)

Answer 9

flank each transposable element insertion

Question 10

Autonomous DNA transposons

Answer 10

-encode their own functional transposase
-have intact ITRs
-able to transpose by themselves

Question 11

Nonautonomous DNA Transposons

Answer 11

-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

Question 12

How do transposons move?

Answer 12

-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

Question 13

Retrotransposons

Answer 13

-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

Question 14

Structural Elements Retrotransposons

Answer 14

-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

Question 15

SINES (short interspersed elements)

Answer 15

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

Question 16

Middle Repetitive DNA

Answer 16

Interspersed Transposons (SINES and LINES)

Question 17

LINES (long interspersed elements)

Answer 17

1-6kb in length; repeated ~850,000x in the human genome
21% of genome
example: the L1 family of DNA sequences

Question 18

Transposable Elements in Disease

Answer 18

-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

Question 19

Proofreading

Answer 19

-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

Question 20

Mismatch Repair

Answer 20

-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

Question 21

Clinical Connection to DNA repair

Answer 21

-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

Question 22

Base Excision Repair

Answer 22

-repairs damage to individual bases on a single DNA strand
*alkylation or other chemical modifications

Question 23

3 Basic Steps to Base Excision Repair

Answer 23

1. DNA glycosylases cut the glycosidic bond b/w target base and its sugar
2. AP* endonuclease cuts the phosphodiester backbone
3. DNA polymerase and ligase fill in the gap & seal it

Question 24

Nucleotide-Excision Repair

Answer 24

-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!

Question 25

Clinical Connection to DNA repair 2

Answer 25

xeroderma pigmentosum

rare recessive disorder

predisposes affected individuals to severe skin abnormalities, skin cancers, & other developmental / neurological defects

Question 26

Double Strand Breaks

Answer 26

-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

Question 27

Homologous Recombination Repair (HRR)

Answer 27

-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

Question 28

Non-Homologous End Joining (NHEJ)

Answer 28

-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