ABSTRACTHMOX1 is an important gene in biosynthesis of the eggshell pigment of blue eggs. Previous studies found that HMOX1 is highly expressed in the shell gland of hens laying blue eggs (BlueH) compared with hens laying brown eggs (BrownH); however, the reasons for the differential expression are unclear. In this study five single nucleotide polymorphism (SNP) in HMOX1 were genotyped in 111 BlueH and 115 BrownH. The association of haplotypes of these SNP with the blue egg phenotype was tested. Haplotype-specific expression of HMOX1 was detected in the shell gland. The interaction of sequence variants and transcription factors was analyzed using electrophoretic mobility shift assay (EMSA). A TG haplotype covering upstream 1.4 kb region of HMOX1 was significantly associated with blue eggs (p<0.05). Furthermore, the birds (n=12) with the haplotype expressed 3.8 fold more transcripts than those (n=12) without the haplotype (p<0.05). After re-sequencing a 2.2 kb region harboring the TG haplotype, a total of 26 SNP were found, of which a SNP was predicted to create a binding site of Nrf2, a transcription factor initiating HMOX1 expression. However, subsequent EMSA failed to confirm the Nrf2-DNA interaction. Taken together, the data suggested that the TG haplotype is not directly involved in regulation of HMOX1 expression; a regulatory mutation located near the haplotype and linked with the haplotype may exist and be responsible for the differential expression ofHMOX1.

ABSTRACT DNA polymerase eta (Pol η) is one of several Y family translesion synthesis (TLS) polymerases in humans and plays an important role in maintaining genome stability after ultraviolet (UV) irradiation, as it carries out error-free TLS at sites of UV-induced lesions. We performed site-directed mutagenesis of human polymerase η gene (Y52E), confirmed by sequencing, then cloned wild-type mutant and POLH genes into the eukaryotic vector pEGFP-N1. After transfecting wild-type and mutant plasmids into HaCaT keratinocytes, we tested for UV-induced cis-syn cyclobutane pyrimidine dimer (CPDs) DNA lesions, and analysed cellular viability by MTT cell proliferation assay. The results showed that CPD levels decreased with empty vector control (EVC), wild-type POLH and Y52E-POLH over 48 hours post-UV irradiation with 0.1 mW/cm2 UVB for 15 minutes (p = 0.025). The rate in CPD reduction of mutant POLH was less than in wild-type POLH. Cell viabilities of all three groups increased over 48 hours after UV irradiation, with the increased rate in the wild-type being higher than for mutant protein (p = 0.046). We conclude that Y52E POLH has reduced capacity to bypass UV-induced DNA lesions in HaCaT cells.

RESUMEN La ADN polimerasa eta (Pol η) es una de las varias polimerasas de la familia Y en los seres humanos, que hace posible la síntesis de translesión (TLS), y desempeña un papel en el mantenimiento de la estabilidad del genoma luego de una radiación UV, ya que lleva a cabo TLS libre de errores en los sitios de las lesiones inducidas por rayos UV. Realizamos mutagénesis de sitio dirigido, del gen η (Y52E) de la polimerasa humana, confirmada por secuenciación, y entonces clonamos genes POHL y genes mutantes de tipo salvaje, transformándolos en un vector eucariótico pEGFP-N1. Después de transfectar los plásmidos de tipo salvaje y mutantes en queratinocitos HaCaT, realizamos pruebas buscando las lesiones dímero de pirimidina cis-syn ciclobutano (CPD) del ADN, inducidas por UV, y analizamos la viabilidad celular mediante ensayo de proliferación de células MTT. Los resultados mostraron que los niveles de CPD disminuían con el control de vector vacío (EVC), el gen POHL de tipo salvaje, y el POHL Y52E, en 48 horas de post irradiación de UV con 0.1 mW/cm2 UVB durante 15 min (p = 0. 025). La tasa de reducción del CPD del mutante POHL fue menor que en el POHL de tipo salvaje. Las viabilidades celulares de los tres grupos aumentaron a las 48 horas después de la irradiación con UV, con un incremento de la tasa del tipo salvaje con respecto de la proteína mutante (p = 0.046). Concluimos que el POHL Y52E ha reducido la capacidad para reparar por bypass replicativo las lesiones del ADN inducidas por la luz ultravioleta en células HaCaT.