Biochemical Features Of The Recq4 Helicase

A. Structural Similarities to Other RecQ Helicase Family Members

The RECQL4 helicase has a helicase domain that contains seven consensus motifs in the middle of the molecule (see Fig. 2). The amino acid sequence of the helicase domain is 40.8% homologous to that of the E. coli RecQ helicase, the prototype of this family, within the same range as the other four family members, RECQL, WRN, BLM, and RECQ5 helicases. Of the five human RecQ helicases, defective WRN, BLM, and RECQL4 helicases are related to diseases that show phenotypes of genomic instability and a high risk of cancer. Because RecQ heli-case-deficient mutants of E. coli and yeast show genomic instability and a high level of recombination, it is conceivable that the function(s) of the RecQ helicase family is conserved across the species and participates in maintaining genomic integrity. In fact, the yeast Sgsl helicase suppresses illegitimate recombination, and the human BLM, WRN, or RECQL4 helicase can substitute this function in the sgsl -defective yeast mutant (74) (Kitao et al., unpublished results).

The WRN helicase contains an exonuclease domain at the N-terminal region of the molecule, and both WRN and BLM helicases have distinct nuclear localization signals (NLS) consisting of basic amino acid arrays at the C-terminal region (71-73). However, the RECQL4 helicase does not contain an exonuclease domain, nor does it contain a clear NLS, although the RTS helicase is transported to the nucleus and is distributed in the nucleolus and the nucleoplasm (75) (see Sec. V.D). These findings suggest that each helicase in the RecQ family has evolved from a progenitor helicase and contains diverged structural features even though its enzymatic action that hydrolyzes ATP and unwinds DNA remains the same. The WRN and BLM helicases are intact in RTS patients with defective RECQL4-defective patients (and conversely WRN patients have intact RECQL4 helicase (Kitao et al., unpublished results), suggesting that these helicases have specific roles and are unable to compensate for one another's functions. In remains to be clarified in what kind of DNA structures these helicases unwind, or with what kind of nuclear factors they cooperate (or both), and in what kinds of cellular events they function.

The RecQL4 helicase has high amino acid homologies to the homologue he-licase in Drosophila (41) and in the mouse (76) (Kitao et al., unpublished results): They have homologies of 53.9% and 78.2% in the helicase domain, respectively. Particularly, the mouse RecQL4 helicase is very similar to that of humans, showing an overall homology as high as 60.7%, whereas the homologies of the Drosophila homolog are restricted to the short N-terminal end (34.0%), the helicase domain (53.9%), and the C-terminal region (32.1%). These data imply that two domains, the helicase and the C-terminal domains, are conserved among species and may be important for the RecQ4 protein's enzymatic activity.

B. Promoter and Tissue-Specific Expression of RECQL4

The upstream region of the open reading frame of RECQL4 contains 1 Sp1 and several AP2 transcription regulatory element sites and no TATA-box near the capping site (Fig. 5), suggesting that the expression of RECQL4 is probably regulated by a housekeeping-type promoter similar to the expression of WRN. Previously, Yamabe and coworkers (77) extensively investigated the expression of WRN and showed that WRN is mainly regulated by Sp1, modulated by tumor-suppressor proteins p53 and Rb, downregulated by p53, and slightly upregulated by Rb. A similar (but not an identical) transcriptional control is predicted to occur with RECQL4 gene expression. In general, the transcription of RECQL4, WRN, and BLM, is upregulated in rapidly growing cells, such as SV40-transformed fibroblasts and EBV-infected lymphoblastoid cells, but no or only a very low level of transcription is observed for all these genes in senescent or resting cells (64).

The tumor-promoting PMA rapidly stimulates the synthesis of RECQL4, WRN, and BLM helicases in resting peripheral B cells; apparently mediated by simultaneous activation of transcription and translation (64). This stimulatory effect of PMA is different in the three helicase genes. PMA stimulates RECQL4 and WRN synthesis in a much shorter period (within approximately 5 hr) than with BLM (within approximately 40 hr). These findings prompt us to speculate that the transcription of RECQL4, as a housekeeping gene, is regulated by a cell cycle similar to WRN.

To determine if each helicase can be expressed in specified tissues or organs, we analyzed the expression of all five RecQ helicase genes in various human organs and tissues by Northern blotting (Fig. 6). The data show that RECQL4, WRN, and BLM are expressed in a tissue specific-fashion, and support the idea that a close correlation exists between the site of gene expression and the site (tissue or organ) of disease phenotypes. For example, WRN is expressed highly in pancreas, testis, and ovary, and WS patients often show clinical symptoms of diabetes and hypogonadism. Also, BLM is highly expressed in the thymus

Figure 5 Potential promoter region of RECQL4 adjacent to the transcription initiation sites. The first intron and the upstream region of RECQL4 contained a high content of GC residues and no TATA box, characteristics of the promoter of a housekeeping gene. The high expression of RECQL4 in the testis and thymus may be explained by the enriched Sp1 transcription factor in the testis and by Sp1 and CREB in thymocytes.

Figure 5 Potential promoter region of RECQL4 adjacent to the transcription initiation sites. The first intron and the upstream region of RECQL4 contained a high content of GC residues and no TATA box, characteristics of the promoter of a housekeeping gene. The high expression of RECQL4 in the testis and thymus may be explained by the enriched Sp1 transcription factor in the testis and by Sp1 and CREB in thymocytes.

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