Base excision repair of oxidative DNA damage coupled with removal of a CAG repeat hairpin attenuates trinucleotide repeat expansion

Trinucleotide repeat (TNR) expansion is responsible for numerous human neurodegenerative diseases. However, the underlying mechanisms remain unclear. Recent studies have shown that DNA base excision repair (BER) can mediate TNR expansion and deletion by removing base lesions in different locations of a TNR tract, indicating that BER can promote or prevent TNR expansion in a damage location–dependent manner. In this study, we provide the first evidence that the repair of a DNA base lesion located in the loop region of a CAG repeat hairpin can remove the hairpin, attenuating repeat expansion. We found that an 8-oxoguanine located in the loop region of CAG hairpins of varying sizes was removed by OGG1 leaving an abasic site that was subsequently 5′-incised by AP endonuclease 1, introducing a single-strand breakage in the hairpin loop. This converted the hairpin into a double-flap intermediate with a 5′- and 3′-flap that was cleaved by flap endonuclease 1 and a 3′-5′ endonuclease Mus81/Eme1, resulting in complete or partial removal of the CAG hairpin. This further resulted in prevention and attenuation of repeat expansion. Our results demonstrate that TNR expansion can be prevented via BER in hairpin loops that is coupled with the removal of TNR hairpins

AP endonuclease 1 prevents trinucleotide repeat expansion via a novel mechanism during base excision repair

Base excision repair (BER) of an oxidized base within a trinucleotide repeat (TNR) tract can lead to TNR expansions that are associated with over 40 human neurodegenerative diseases. This occurs as a result of DNA secondary structures such as hairpins formed during repair. We have previously shown that BER in a TNR hairpin loop can lead to removal of the hairpin, attenuating or preventing TNR expansions. Here, we further provide the first evidence that AP endonuclease 1 (APE1) prevented TNR expansions via its 3′-5′ exonuclease activity and stimulatory effect on DNA ligation during BER in a hairpin loop. Coordinating with flap endonuclease 1, the APE1 3′-5′ exonuclease activity cleaves the annealed upstream 3′-flap of a double-flap intermediate resulting from 5′-incision of an abasic site in the hairpin loop. Furthermore, APE1 stimulated DNA ligase I to resolve a long double-flap intermediate, thereby promoting hairpin removal and preventing TNR expansions

A 5\u27, 8-cyclo-2\u27-deoxypurine lesion induces trinucleotide repeat deletion via a unique lesion bypass by DNA polymerase β.

5′,8-cyclo-2′-deoxypurines (cdPus) are common forms of oxidized DNA lesions resulting from endogenous and environmental oxidative stress such as ionizing radiation. The lesions can only be repaired by nucleotide excision repair with a low efficiency. This results in their accumulation in the genome that leads to stalling of the replication DNA polymerases and poor lesion bypass by translesion DNA polymerases. Trinucleotide repeats (TNRs) consist of tandem repeats of Gs and As and therefore are hotspots of cdPus. In this study, we provided the first evidence that both (5′R)- and (5′S)-5′,8-cyclo-2′-deoxyadenosine (cdA) in a CAG repeat tract caused CTG repeat deletion exclusively during DNA lagging strand maturation and base excision repair. We found that a cdA induced the formation of a CAG loop in the template strand, which was skipped over by DNA polymerase β (pol β) lesion bypass synthesis. This subsequently resulted in the formation of a long flap that was efficiently cleaved by flap endonuclease 1, thereby leading to repeat deletion. Our study indicates that accumulation of cdPus in the human genome can lead to TNR instability via a unique lesion bypass by pol β

Crosstalk between MSH2–MSH3 and polβ promotes trinucleotide repeat expansion during base excision repair

Studies in knockout mice provide evidence that MSH2–MSH3 and the BER machinery promote trinucleotide repeat (TNR) expansion, yet how these two different repair pathways cause the mutation is unknown. Here we report the first molecular crosstalk mechanism, in which MSH2–MSH3 is used as a component of the BER machinery to cause expansion. On its own, pol β fails to copy TNRs during DNA synthesis, and bypasses them on the template strand to cause deletion. Remarkably, MSH2–MSH3 not only stimulates pol β to copy through the repeats but also enhances formation of the flap precursor for expansion. Our results provide direct evidence that MMR and BER, operating together, form a novel hybrid pathway that changes the outcome of TNR instability from deletion to expansion during the removal of oxidized bases. We propose that cells implement crosstalk strategies and share machinery when a canonical pathway is ineffective in removing a difficult lesion

Simulator for Virtual Instrumentation Courses

Import 05/08/2014Tato bakalářská práce se zabývá návrhem a realizací simulátoru pro výuku virtuální instrumentace. Tento simulátor je zkonstruován na bázi mikrokontroléru, který obstarává hlavní funkčnost celého zařízení a je naprogramován tak, aby uměl vykonávat funkce napětím řízeného oscilátoru, napětím řízeného PWM, simulátoru servomotoru a generátoru impulzů. Částí této práce je i vytvoření řídícího programu, který komunikuje s mikrokontrolérem pomocí sériové linky a umožňuje parametrizaci a nastavení funkce simulátoru.This bechelor thesis describes the design and implementation of simulator for the teaching of virtual instrumentation courses. This simulator is based on microcontroller, which performs the main functionality of the device and is programmed to be able to perform the functions of a voltage-controlled oscillator, voltage controlled PWM, the simulator of servo motor and a pulse generator. Part of this work is the creation of a application that communicates with a microcontroller via a serial line and allows the configuration and settings of the simulator.450 - Katedra kybernetiky a biomedicínského inženýrstvívelmi dobř