Ch 14: Site-specific recombination and transposition

general homologous recombination

recombination between two identical/very similar sequences

site-specific recombination

recombination limited to specific sequences

recombinase

• binds to specific recognition sites (20-200 bp with inverted repeats on both sides of unique core sequences)

• catalyzes site-specific recombination

transposition

• transposons (short DNA segment) can transpose (move) to different location within the genome

• they can also leave a copy in original location in some cases

transposon

• DNA segment that is replicated/moved to different location within the genome

• either within chromosome or to a different chromosome

• can move with or without leaving copy in original location

retrotransposons

• RNA segment that is reverse transcribed to DNA then replicated/moved to different location within the genome

• either within chromosome or to different chromosome

• can move with or without leaving copy in original location

site-specific recombination recognition sites

• loxP or FRT are examples of ______

• has palindromic inverted repeats with nonpalindromic core sequence in the middle

inversion (FRT—> eneg <—FRT)

• opposite orientation of 2 recognition sites leads to ____ of the gene after site-specific recombination

• FRT—> gene <— FRT

deletion (FRT—> + circular FRT—>gene)

• same orientation of 2 recognition sites leads to ____ of the gene after site-specific recombination

• FRT—> gene —>FRT

recombinase

• acts like restriction enzyme + DNA ligase

• cleaves at specific recognition sites and seals double stranded breaks after cleaving

phosphodiester

recombinase doesn’t require ATP because ______ bond energy is conserved in DNA protein bond

Tyr (tyrosine)

recombinases using an active site ___ for cleavage cut ONE strand in each homologous pair at a time producing a Holiday intermediate

Ser (serine)

recombinases using an active site ____ cut BOTH pairs of recognition sites at the same time → no Holiday intermediate

site-specific recombination

• interlinked genomes as a result of issues during normal replication can be separated using ________

• Holiday structures that are intertwined during DSB repair

• catenated circular DNA during replication

site-specific recombination

• can be used by phage and plasmids during rolling circle replication

• make replication forks move in the same direction around plasmid

lambda phage

• site-specific recombination is used by _____ to integrate into bacterial genome

• lytic: goes in, replicates, comes out

• lysogenic: goes in, integrates, ride along in bacterial genome

circularize

site-specific recombination is used by phage P1 to ____ genome and during DNA replication (rolling circle)

Hin

bacterial recombinase that inverts promoter segment every 1000 generations (phase shift) in order to evade host immune system

phage P1

• Cre-Lox system from ______

• Cre recombinase (causes recombination)

• LoxP recognition site (location of crossover)

conditional gene knockouts

promotor that controls Cre recombinase transcription ensures that it is only transcribed in the target gene tissue

Cre Lox FLEx vector switches

uses loxP with mutant core to switch gene expression on and off using Cre recombinase

inactive

• loxP —> lox2272—> eneg <— loxP <—lox2272

• initial configuration for Cre Lox FLEx vector switches

• the gene is ____ because it is inverted

active

• loxP —> lox2272—> eneg <— loxP <—lox2272

• to

• loxP —> gene <—lox2272 <— loxP <—lox2272

• first inversion of loxP (could be either lox sites) for Cre Lox FLEx vector switches

• the gene is ____ because it is not inverted

permanently, deleted

• loxP —> gene <—lox2272 <— loxP <—lox2272

• to

• loxP —> gene <—lox2272

• second inversion of lox2272 now in same direction for Cre Lox FLEx vector switches

• the gene is ____ active because one loxP and one lox2272 were ____

inducible promoter

• gene of interest only flipped (Cre only transcribed) when inducer is present

• ex) Tamoxifen is inducer (estrogen receptor modulator)

double strand break, 3’ OH

in cut and paste transposition, original copy gets _____ and transposon is excised → _____ attacks and inserts into new location

single strand breaks, 3’ OH, Holiday structure

in replicative transposition, original copy gets ____ and transposon’s _____ attacks and inserts into new location forming _____ then replication and repair happens

reverse transcribed, retrotransposon, 3’ OH

only the transposable element of DNA is transcribed into RNA and then _____ into the _______ then its _____ attacks and inserts into new location

transposase, 3’ OH

• ______ cleaves each side of transposon to expose ____ → which attacks opposite strand forming a hairpin loop and excising the transposon

• cut and paste transposition

transposase, 3’ OH

• _____ cleaves hairpins on transposon after excision to re-expose _____ to attack target DNA

• cut and paste transposition

transposase, 3’ OH, Holiday-like structure

• both strands of donor DNA are cleaved by ______ on opposite sides of the transposon, revealing only the transposon’s ______, which attacks the target DNA, forming a ______

• replicative transposition

cointegrate (like holiday structure), recombination (homologous or site-specific)

• after forming the Holiday-like structure, the transposon is replicated to create a ______, which is resolved by _______

• replicative transposition

composite transposon (Tn5)

two transposons with some other DNA sequence in between

complex transposon (Mu)

transposon that includes other transposition regulatory genes besides transposase and resistance genes

effects of transposition

• allow antibiotic resistance genes to spread between bacterial populations

• inversions, deletions, translocations

• change in regulatory sequences or chromosome structure

• produce different gene product

selfish DNA

transposons referred to as _____ because they exist without any benefit to host

transposition

production of inhibitor proteins or short inhibitory RNA (RNAi) accumulates and controls ______

hybrid dysgenesis

• P strain male x M strain female in Drosophila → sterile offspring due to highly active (unregulated) transposons

• P strain = has active transposable elements (TE)

• M strain = lacks TE and doesn’t have system (inhibitor) to repress it

mother

• transposon inhibitors only inherited from ____

• P female x M male cross → fertile offspring that carry P element

• P male x M female cross → sterile offspring that didn’t inherit inhibitors to repress transposition

retrotransposons

long interspersed elements (LINES 1-6 kbp),

short interspersed elements (SINES 150-500 bp), and

long terminal repeats (LTR 6-9 kbp)

are examples of ____

Mariner elements

• potentially transpose in a wide range of species via cut and paste

• most are inactive due to mutations in transposase gene

44%

transposable elements make up ___ of the human genome

CD4 and chemokine

• retroviral infection requires two receptors: ___

• some people with altered receptors are partially resistant to HIV

retroviruses

• HIV, and Rouse Sarcoma virus are examples of ______

• people with altered receptors can be partially resistant to HIV

promoter region

• region of retrovirus genome not transcribed into mRNA by RNA pol II

• if this region isn’t transcribed then it can’t regulate LTRs (transposable elements)

reverse transcriptase

• slow and error prone

• 1 error per 10⁴ to 10⁶ bases (1 mistake every time genome is copied)

• DNA pol II error rate: 1 error per 10⁶ to 10⁸

0.6 bp/sec

• speed of reverse transcriptase

• 4 hours to copy 9 kbp genome

50 bp/sec

speed of human DNA pol

1000 bp/sec

speed of E.coli DNA pol III

gag

virion (structural) proteins in retroviruses

pol

reverse transcriptase, integrase protein in retroviruses

env

envelope proteins in retroviruses

one mRNA

gag (structural), pol (RT), and env (envelope) proteins are transcribed from ____

reading frame shift, pseudoknot

gag-pol-env are in same mRNA but in order to transcribe pol or env there needs to be a _____ caused by a _____

alternative splicing

gag-pol-env are in same mRNA but in order to produce env protein, gag and pol regions of mRNA are removed by _____

viable viruses

• transducing viruses that have some other gene in the place of env can infect but are unable to form ____

• could add “helper virus” that could make envelope proteins

8%

LTR retrotransposons make up ___ of the human genome

virus-like-particles (VLP)

LTR transposons form _____ which can’t leave the cell

AZT

retrovirus reverse transcriptase inhibitor

Paxlovid

retrovirus protease inhibitor

antibody structure

2 identical light (L) chains + 2 identical heavy (H) chains each with variable (V) domain and constant domain

recombination signal sequences (RSS)

• used for somatic recombination

• flanked by V and J segments of antibodies

allelic exclusion

• each B cell (white blood cell that makes antibodies) expresses only one type of antibody even though it has 2 homologous sets of chromosomes

• successful rearrangement which produces active light/heavy chain suppresses further rearrangements

class switching

• structural requirements on constant part of antibody changes during immune response

• allows B cell to change the antibody it produces without altering antigen specificity