L8: Tandem repeats & Repeat expansion disorders Flashcards
What makes up the majority of our genome?
Repetitive DNA that includes TEs and related sequences (44%)
What are the two main categories of repeated elements?
tandem repeats and dispersed repeats
What are the three types of tandem repeats?
Tandem paralogues
Satellite DNA
rDNA
What are the three types of satellite DNA?
(Macro)Satellites
Minisattelites
Microsattelites
What are the four types of dispersed repeats?
Paralogues
Transposons
tDNAs
Retro(pseduo-)genes
How can transposons be further categorised?
Class I:
LINEs
SINEs
LTR retrotransposons
Class II:
DNA transposons
What is the difference between the different satellites?
Their motif length:
(Macro)Satellites: (>100bp)
Minisattelites (10- 100bp)
Microsattelites (1-9bp)
What is the max length of satellite DNA?
They are very large arrays of repetitive DNA – each repeat typically kilobases long. Satellite DNA can extend over megabases of DNA but its maximum length is unknown
Give some other names for tandem repeats
Variable number tandem repeats VNTR (most frequently used when referring to minisatellites)
- Simple repeat
- Short tandem repeat (STRs)
- Simple sequence repeats (SSRs)
(most frequently used when referring to microsatellites)
Where are macro satellites most commonly found?
They are most commonly found at centromeres and in heterochromatin (cytologically dense material that is typically found at centromeres and telomeres)
Typically, how long are minisatellites and how often do they repeat?
Range in length from 10-60 base pairs, typically repeated 5-50 times.
How many minisatelites are there in the human genome?
More than 1000 locations in the human genome
How variable are minisatellites?
Highly variable between individuals in terms of repeat length
Why would a sequence be classified as a mini satellite?
Repeat sequence is 10-100, repeats 5-50 times and the number of repeats varies between people but the motif itself does not.
Typically, how long are microsatellites and how often do they repeat?
- Motifs range from one to ~six base pairs
- Motif typically repeated 5-50 times
What are the most common motif lengths for microsattelites and why?
Often 3-6 so no frame shift
How variable are microsatellites?
- Very high mutation rate compared to other regions in the genome
- Highly variable between individuals
What are the consequences of this variability in microsatellies?
Often pathological
In what ways can tandem repeats vary
- Tandem repeats are highly variable in the number of repeat copies
- They vary between individuals but can also vary within an individual – for example in different cell types or tissues
When was DNA fingerprinting first developed and how was it done?
Developed in the 1980s, Originally used restriction enzymes to fragment DNA and then a southern blot to detect fragment length
What do modern DNA fingerprinting techniques utilise?
PCR
What structural effects can short tandem repeats have on DNA?
STRs are able to form secondary DNA structures such as G quadruplexes. Tandem repeats are very complimentary and are likely to repeat these secondary structures
G-quadruplex (G4) structures are only one of many (ten or more) non-B-form DNA secondary structures analysed to date. Briefly describe three well-studied structures
Z-DNA: In contrast to standard B-form DNA (B-DNA), Z-DNA is a left-handed helix. Z-DNA motifs (that is, sequences that form Z-DNA in vitro) are tracts of alternating purines and pyrimidines. Negative supercoiling stabilizes the formation of Z-DNA under physiological salt conditions130, and it is hypothesized that Z-DNA relieves transcription-induced torsional stress
Cruciform structures: Negative supercoiling can also cause B-DNA to adopt a four-armed, cruciform secondary structure. These structures require ≥6-nucleotide inverted repeats (cruciform motif) to form, and such motifs are located near replication origins, breakpoint junctions and promoters in diverse organisms
Triplex DNA: Three-stranded triplex DNA occurs when single-stranded DNA forms Hoogsteen hydrogen bonds in the major groove of purine-rich double-stranded B-DNA. Triplexes in which the third strand is antiparallel to the DNA duplex can form at physiological pH, and these structures are stabilized by negative supercoiling
What secondary structures can form in CAG, CTG, and CCG repeats?
Hairpins of the As Ts or Cs, meaning in this structure they have no pairing. This can happen in both odd and even repeats although the pattern differs slightly in odd repeats.
What secondary structure can form in GAA repeats?
Triple helice formed by (GAA)n repeats
What secondary structure can form in CCG repeats?
Tetraplex structures; G4s- unwinding this structure takes a lot of force
Name three models for STR repeat expansion
- Replication slippage model
- Double strand break repair model
- Transcription mediated model
Describe the replication slippage model
During replication of a repeat-containing sequence, the replication machinery may pause on the lagging strand, due to secondary structures or other kinds of lesions.
Partial unwinding of the lagging strand may lead to replication slippage when replication restarts, giving rise to an expansion or a contraction of the repeat tract, depending on what strand (template or newly synthesised strand) slippage occurred.
Alternatively, partial unwinding of the lagging strand may lead to lesion bypass by homologous recombination with the sister chromatid, also leading to contractions or expansions of the repeat tract
Describe the double stranded break model
Following a DSB, gene conversion is initiated by strand invasion, forming a “D-loop.” (two strands of a double-stranded DNA molecule are separated for a stretch and held apart by a third strand of DNA)
DNA synthesis within the repeat tract may be faithful or associated with slippage. After capture of the second end of the break, DNA synthesis of the second strand may also be faithful or associated with slippage.
Slippage events will lead to expansions of the repeat tract or to contractions if slippage occurs on the template strand.
Alternatively, after capture of both ends followed by DNA synthesis, the two newly synthesized strands may unwind and anneal with each other in frame or out of frame, leading to expansions or contractions of the repeat tract, however it is more biased to expansion than contraction.
Describe transcription mediated repair
Transcription through CAG·CTG repeats promotes the formation of slipped-strand structures, which subsequently stall RNA polymerase (RNAP) and lead to recruitment of the nucleotide excision repair (NER) machinery. Transcription-coupled NER removes the portion of the transcribed strand containing the RNAP-blocking hairpin; the resulting gap is filled in using the non-transcribed strand (NTS) as a template. Depending on the location of loops on the NTS relative to the removed hairpin, the repair event will either expand or contract the trinucleotide repeat.
Why are STRs difficult to study using PCR?
The underlying properties of STRs make them difficult to study using PCR based methods, they show up in laddering fragments during gel electroporesis
